Il-6 antagonists and uses thereof

ABSTRACT

IL-6 antagonists are provided that are specific for binding to site II of IL-6. Methods of using such inhibitors to treat IL-6 related diseases, e.g., disease of the eye such as diabetic macular edema are disclosed.

RELATED APPLICATIONS

The present application is a continuation-in-part of InternationalApplication No. PCT/US2013/069279, filed on Nov. 8, 2013, which claimspriority to U.S. Application Ser. No. 61/723,972, filed Nov. 8, 2012 andU.S. Application Ser. No. 61/831,699, filed Jun. 6, 2013. The entirecontent of each of the foregoing applications is hereby incorporatedherein by reference.

FIELD OF THE INVENTION

The field of the invention relates to IL-6. More particularly, the fieldrelates to modulators of IL-6 and their uses in treating disease suchdiseases of the eye.

BACKGROUND

IL-6 is a pleiotropic cytokine with reported roles in inflammation,hematopoiesis, angiogenesis, cell differentiation, and neuronalsurvival.

SUMMARY

The invention relates to IL-6 antibodies and fragments, and derivativesof such antibodies and fragments that have certain features, includingbinding specificity to site II of IL-6, and methods of using suchantibodies, fragments, and derivatives. Accordingly, the presentinvention relates to an antibody, fragment, or derivative thereof thatcan specifically bind to site II of an IL-6. In some embodiments, theantibody, fragment, or derivative can bind to IL-6 with a K_(D) of 240pM or less. In some embodiments, the antibody, fragment, or derivativehas a T_(m) of 70° C. or greater. In some embodiments, the antibody,fragment, or derivative can bind to IL-6 with a K_(D) of 240 pM or lessand has a T₅₀ of 70° C. or greater. The antibody or fragment orderivative thereof can, in some cases, bind to at least one of R24, K27,Y31, D34, S118, or V121 of human IL-6; in some cases the antibody orfragment or derivative thereof can bind to R24, K27, Y31, D34, S118, andV121 of human IL-6. In embodiments, the antibody or fragment orderivative thereof can bind to at least 1, at least 2, at least 3, atleast 4, or at least 5 of R24, K27, Y31, D34, S118, and V121 of humanIL-6.

In one aspect provided herein is an antibody or fragment thereof (e.g.,an antigen binding fragment thereof) that can specifically bind to siteII of a human IL-6.

In embodiments, the antibody or fragment thereof (e.g., an antigenbinding fragment thereof) can bind to an IL-6 with a K_(D) of 200 pM orless.

In embodiments, the antibody or fragment thereof (e.g., an antigenbinding fragment thereof) can bind to an IL-6 with a K_(D) of 200 pM orless and/or has a T_(DB) of 70° C. or greater.

In embodiments, the antibody or fragment thereof (e.g., an antigenbinding fragment thereof) can bind to an IL-6 with a K_(D of) 200 pM orless and/or has a T_(DB) of 80° C. or greater.

In embodiments, the antibody or fragment thereof (e.g., an antigenbinding fragment thereof) binds to at least one of R24, K27, Y31, D34,S118, and V121 of a human IL-6. In embodiments, the antibody or fragmentthereof (e.g., an antigen binding fragment thereof) binds to at leasttwo of R24, K27, Y31, D34, S118, and V121 of a human IL-6. Inembodiments, the antibody or fragment thereof (e.g., an antigen bindingfragment thereof) binds to at least three of R24, K27, Y31, D34, S118,and V121 of a human IL-6. In embodiments, the antibody or fragmentthereof (e.g., an antigen binding fragment thereof) binds to at leastfour of R24, K27, Y31, D34, S118, and V121 of a human IL-6. Inembodiments, the antibody or fragment thereof (e.g., an antigen bindingfragment thereof) binds to at least five of R24, K27, Y31, D34, S118,and V121 of a human IL-6. In embodiments, the antibody or fragmentthereof (e.g., an antigen binding fragment thereof) binds to R24, K27,Y31, D34, S118, and V121 of human IL-6.

In an aspect provided herein is an an antibody (e.g., an isolatedantibody, e.g., an isolated monoclonal antibody) or a fragment thereof(e.g., an antigen binding fragment thereof) that comprises (a) a VH CDR1as set forth in SEQ ID NO:4, a VH CDR2 as set forth in SEQ ID NO:5, andVH CDR3 as set forth in SEQ ID NO:6; and optionally (b) a VL CDR1 as setforth in SEQ ID NO:7, a VL CDR2 as set forth in SEQ ID NO:8, and a VLCDR3 as set forth in SEQ ID NO:9, wherein the antibody or fragmentthereof (e.g., the antigen binding fragment thereof) can specificallybind to a human IL-6. In embodiments, the antibody or fragment thereofcomprises heavy chain CDRs VH CDR1, VH CDR2, and VH CDR3 that areidentical to the CDR sequences respectively set forth in SEQ ID NO:4,SEQ ID NO:5 and SEQ ID NO:6 or that collectively differ from said CDRsequences by no more than 1, 2, 3, 4, or 5 amino acids. In embodiments,the antibody or fragment thereof comprises light chain CDRs, VL CDR1, VLCDR2, and VL CDR3 that are identical to the CDR sequences respectivelyset forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 or thatcollectively differ from said CDR sequences by no more than 1, 2, 3, 4,or 5 amino acids.

In an aspect provided herein is an IL-6 antibody (e.g., an isolated IL-6antibody) or a fragment thereof (e.g., an antigen binding fragmentthereof) that can dissociate from human IL-6 with a K_(D) of 240 pM orless (e.g., as determined by surface plasmon resonance). In embodiments,the antibody can neutralize IL-6 activity with an IC50 of 255 pM orless, e.g., as determined in an in vitro HEK-Blue ™ IL-6 assay.

In an spect provided herein is an IL-6 antibody ((e.g., an isolated IL-6antibody) or a fragment (e.g., an antigen binding fragment) thereof,that can competitively inhibit binding to a human IL-6 by an antibody orfragment (e.g., an antigen binding fragment) thereof comprising SEQ IDNO:1 and SEQ ID NO:2 or an antibody or fragment (e.g., an antigenbinding fragment) thereof comprising SEQ ID NO:3 and SEQ ID NO:2.

In embodiments, the antibody or fragment thereof (e.g., an antigenbinding fragment thereof) has a monovalent Kd of 2 nM or greater at pH5.5.

In embodiments, the antibody or fragment thereof (e.g., an antigenbinding fragment thereof) is an IgG2 antibody.

In embodiments, the antibody or fragment thereof (e.g., an antigenbinding fragment thereof) has altered FeRn binding compared to areference antibody. In embodiments, the FeRn binding is decreasedcompared to a reference antibody. In embodiments, the Fc domain of theantibody or fragment is altered compared to a reference antibody.

In an aspect provided herein is an IL-6 antibody (an antibody that iscapable of binding to an IL-6, e.g., to site II of a human IL-6) orfragment thereof that has a modified Fc domain and exhibits reduced FcRnbinding compared to a corresponding antibody having a wild type Fcdomain.

In embodiments, the antibody or fragment thereof (e.g., an antigenbinding fragment thereof) comprises a mutation at one or more of H311,I254, and H436 of SEQ ID NO:23.

In embodiments, the antibody or fragment thereof (e.g., an antigenbinding fragment thereof) comprises a mutation at two or more of H311,D313, I254, and H436 of SEQ ID NO:23.

In embodiments, the antibody or fragment thereof (e.g., an antigenbinding fragment thereof) comprises a mutation at three or more of H311,D313, I254, and H436 of SEQ ID NO:23.

In embodiments, the antibody or fragment thereof (e.g., an antigenbinding fragment thereof) comprises a mutation at each of H311, D313,I254, and H436 of SEQ ID NO:23.

In embodiments, the antibody or fragment thereof (e.g., an antigenbinding fragment thereof) comprises one or more mutations (e.g., 1, 2,3, or 4 mutations) selected from the group consisting of H311A, H311E,H311N, D313T, I254A, I254R, and H435A.

In embodiments, the antibody or fragment thereof (e.g., an antigenbinding fragment thereof) is isolated.

In embodiments, the antibody is a monoclonal antibody or a fragmentthereof (e.g., an antigen binding fragment thereof). In embodiments, theantibody is a human monoclonal antibody.

In embodiments, the antibody is an isolated monoclonal antibody or afragment thereof (e.g., an antigen binding fragment thereof).

In embodiments, the antibody (e.g., the isolated monoclonal antibody)comprises SEQ ID NO:23.

Also provided herein is an antibody or fragment (e.g., an antigenbinding fragment) thereof (e.g., an IL-6 antibody or fragment thereof asdescribed herein), or a composition comprising such an antibody orfragment thereof, for use in the treatment of an IL-6 associated disease(e.g., for use in the treatment of a subject, e.g. a human subject,having an IL-6 associated disease). In embodiments, said disease is anocular disease characterized by an elevated level of IL-6 in thevitreous. In embodiments, said disease is diabetic macular edema (DME),diabetic retinopathy, uveitis, dry eye disease, uveitis, age-relatedmacular degeneration (AMD), proliferative diabetic retinopathy (PDR),retinal vein occlusion (RVO), neuromyelitis optica (NMO), cornealtransplant, corneal abrasion, or physical injury to the eye. Inembodiments, said disease is DME. In embodiments, said disease is dryeye disease. In embodiments, said disease is dry eye syndrome. Inembodiments, said disease is uveitis. In embodiments, said disease isAMD. In embodiments, said disease id PDR. In embodiments, said diseaseis PDR. In embodiments, said disease is corneal transplant, cornealabrasion, or physical injury to the eye. In embodiments, the antibody orfragment (e.g., the antigen binding fragment) thereof is suitable fordelivery to the vitreous of the eye. In embodiments, the antibody orfragment (e.g., the antigen binding fragment) thereof is delivered tothe vitreous of the eye.

Also provided herein is a method of treating an IL-6 associated disease,the method comprising administering to a subject an IL-6 antibody orfragment thereof (e.g., an antigen binding fragment thereof), e.g., anIL-6 antibody or fragment thereof as described herein. In embodiments,the IL-6 antibody or fragment thereof (e.g., an antigen binding fragmentthereof), is administered in a therapeutically effective amount. Inembodiments, the IL-6 associated disease is an ocular diseasecharacterized by an elevated level of IL-6 in the vitreous. Inembodiments, the IL-6 associated disease is diabetic macular edema(DME), diabetic retinopathy, uveitis, dry eye syndrome, dry eye disease,uveitis, age-related macular degeneration (AMD), proliferative diabeticretinopathy (PDR), retinal vein occlusion (RVO), neuromyelitis optica(NMO), corneal transplant, corneal abrasion, or physical injury to theeye. In embodiments, the antibody or fragment thereof (e.g., the antigenbinding fragment thereof), is suitable for delivery to the vitreous ofthe eye. In embodiments, the antibody or fragment thereof (e.g., theantigen binding fragment thereof), is delivered to the vitreous of thesubject's eye. In embodiments, the IL-6 associated disease is diabeticmacular edema and the antibody or fragment thereof is delivered to thevitreous of the subject's eye.

In another aspect provided herein is a vector comprising a sequenceencoding an IL-6 antibody or fragment thereof (e.g., an antigen bindingfragment thereof) described herein. In embodiments, the vector comprisesa sequence encoding SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, or SEQ ID NO:9.

In another aspect provided herein is a cell that can express thesequence of an IL-6 antibody or fragment thereof (e.g., an antigenbinding fragment thereof) described herein. In embodiments, the cell canexpress one or more of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, or SEQ ID NO:9.

In yet another aspect, the invention relates to a method of reducingsystemic effects of inhibiting an IL-6 in a subject, the methodcomprising administering to the subject an antibody or fragment thereofthat can inhibit an activity of IL-6 and has reduced Fc activitycompared to a corresponding antibody or fragment thereof having a wildtype Fc domain. In some cases, the method of reducing systemic effectsof inhibiting an IL-6 in a subject including administering to thesubject an IL-6 antagonist that has FcRn binding greater than 1 μM,e.g., at low pH such as pH 5.5.

As used herein, the term “antibody” is synonymons with immunoglobulinand is to be understood as commonly known in the art. The term antibodyis not limited by any particular method of producing the antibody. Forexample, the term antibody includes, inter alia, recombinant antibodies,monoclonal antibodies, and polyclonal antibodies. As used herein, anantibody is a tetramer, and unless otherwise disclosed, each is composedof two identical pairs of polypeptide chains, each pair having one lightchain and one heavy chain. The amino terminus of each chain comprises avariable region of about 100 to 120 or more amino acids that play aprimary role in antigen recognition. The carbody-terminal portion ofeach chain comprises a constant region with a primary role in antibodyeffector function. Classes of human light chains are termed kappa andlambda light chains. Heavy chain classes are mn, delta, gamma, alpha, orepsilon, and define the isotype of an antibody. Antibody isotypes areIgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavychains, the variable and constant regions are joined by a “J” region ofabout 12 or more amino acids, with heavy chain also including a “D”region of about three or more amino acids.

The variable regions of each heavy/light chain pair (VH and VL),respectively, form the antigen binding site. Accordingly, an intact IgGantibody, for example, has two binding sites. Except in bifunctional orbispecific antibodies, the two binding sites are the same.

Variable regions of antibody heavy and light chains exhibit the samegeneral structure of relatively conserved framework regions (FR) joinedby three hypervariable regions, also termed complementary determiningregions or CDRs. The term “variable” refers to the fact that certainportions of the variable domains differ extensively in sequence amongantibodies and are involved in the binding and specificity of eachparticular antibody for its particular antigen. Variability liesprimarily in the CDRs, which are separated by the more highly conservedframework regions (FRs). The assignment of amino acids to each domain ismade in accordance with the definitions of Kabat Sequences of Proteinsof Immuological Interest (National Institutes of Heaslth, Bethesda, Md.(1987 and 1991)), or Chothia and Lesk, J Mol Biol 196:901-917 (1987);Chothia et al., Nature 342:878-883 (1989), which describe methods knownin the art.

“Wild type” can refer to the most prevalent allele or species found in apopulation or to the antibody obtained from a non-manipulated animal, ascompared to an allele or polymorphism, or a variant or derivativeobtained by a form of manipulation, such as mutagenesis, use ofrecombinant methods and so on to change an amino acid of theantigen-binding molecule.

The term “antibody fragment” refers to a portion of an intact or afull-length chain or an antibody, generally the target binding orvariable region. Examples of antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)2 and Fv fragments. A “functional fragment”or “analog of an anti-IL-6 site II antibody” is a fragment that canprevent or substantially reduce the ability of IL-6 to bind to receptor,reduce the ability of IL-6/IL-6R complex to bind to gp130, or reduce theability of ligand to bind to gp130 or to initiate signaling. As usedherein, functional fragment generally is synonymous with, “antibodyfragment” and with respect to antibodies, can refer to gragments, suchas Fv, Fab, F(ab′)2 and so on which can prevent or substantially reducethe ability of IL-6 to bind to a receptor, reduce the ability ofIL-6/IL-6R complex to bind to gp130, or to initiate signaling.

A “derivative” of an antibody is a polypeptide that can specificallybind to sit II of IL-6 and shares sequence with an IL-6 site IIantibody, e.g., shares at least one CDR of an antibody that canspecifically bind site II of a human IL-6.

“Compete” means that a first antibody, or fragment thereof can competefor binding with a second antibody or a fragment thereof, such thatbinding of the first antibody with its epitope is detectably decreasesin the presence of the second antibody compared to the binding of thefirst antibody in the absence of the second antibody. In some cases, theterm can also refer to the binding of the second antibody to its epitopewhich is detectably decreased in the presence of the first antibody. Themechanism of such competition can be via, in non-limiting examples,steric hindrance, conformational change, binding to a common epitope.

The term “percent sequence identity” in the context of nucleic acidsequences means the residues in two sequences that are the same whenaligned for maximum correspondence. The length of sequence identitycomparison may be over at least about nine nucleotides, for example, atleast about 18 nucleotides, at least about 24 nucleotides, at leastabout 28 nucleotides, at least about 32 nucleotides, at least about 36nucleotides, or at least about 48 or more nucleotides. Algorithms knownin the art can be used to measure nucleotide sequence identity. Forexample, polynucleotide sequences can be compared using FASTA, Gap orBestfit (Wisconsin Package Version 10.0, Genetics Computer Group (GCG),Madison, Wis.). FASTA, includes, e.g., the programs FASTA2 and FASTA3,provides alignments and percent sequence identity of the regions of thebest overlap between the query and search sequences (Pearson, MethodsEnzymol 183:63-98 (1990); Pearson, Methods Mol Biol 132:185-219 (2000);Pearson, Methods Enzymol 266:227-258 (1996); Pearson, J Mol Biol276:71-84 (1998); incorporated herein by reference). Default parametersfor a particular program or algorithm are typically used. For example,percent sequence identity between nucleic acid sequences can bedetermined using FASTA with its default parameters (a word size of 6 andthe NOPAM factor for the scoring matrix) or using Gap with its defaultparameters as provided in GCG Version 6.1, incorporated herein byreference.

The term “percent sequence identity” in the context of amino acidsequences means the residues in two sequences that are the same whenaligned for maximum correspondence. The length of sequence identitycomparison may be over at least about five amino acid residues, forexample, at least about 20 amino acid residues, at least about 30 aminoacid residues, at least about 50 amino acid residues, at least about 100amino acid residues, at least about 150 amino acid residues, or at leastabout 200 or more amino acid residues. Sequence identity forpolypeptides is typically measured using sequence analysis software.Algorithms for determination of percent sequence identity are known inthe art. For example, amino acid sequences can be compared using FASTA,Gap or Bestfit (Wisconsin Package Version 10.0, Genetics Computer Group(GCG), Madison, Wis.). Protein analysis software matches sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conserative amino acid substitutions. Forexample, GCG contains programs such as “Gap” and “Bestfit,” which can beused with default parameters as specified by the programs to determinesequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and an analog thereof. See,e.g., GCG Version 6.1 (University of Wisconsin, Madison, Wis.).Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, see GCG Version 6.1. FASTA (e.g., FASTA2 andFASTA3) provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences (Pearson,Methods Enzymol 183:63-98 (1990); Pearson, Methods Mol Biol 132:185-219(2000)). Another algorithm that can be used when comparing a sequence toa database containing a large number of sequences from differentorganisms is the computer program BLAST, e.g., blastp or tblastn, usingdefault parameters as supplied with the programs. See, e.g., Altschul etal., J Mol Biol 215:4403-410 (1990); Altschul et al., Nucleic Acids Res25:3389-402 (1997).

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous,” or “substantially purified” when at least about 60 to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein mya be monomeric or multimeric. A substantially pure polypeptideor protein can comprise about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%pure; for example, a substantially pure polypeptide or protein is 50%,60%, 70%, 80%, 90%, 95%, 98%, or 99% pure. Protein purity or homogeneitycan be assessed by an appropriate means, such as polyacrylamide gelelectrophoresis of a protein sample followed by visualizing one or morebands associated with the protein or polypeptide (e.g., upon stainingthe gel), size-exclusion HPLC, cation-exchange HPLC, reduced capillaryelectrophoresis in SDS, peptide mapping, or glycan mapping. Higherresolution can be achieved using methods known in the art, for example,or other means of purification.

The term “substantial similarity” when referring to a nucleic acid orfragment thereof, means that when optimally aligned with appropriatenucleotide insertions or deletions with another nucleic acid (or itscomplementary strand), there is nucleotide sequence identity in at leastabout 85%, at least about 90%, and at least about 95%, 96%, 97%, 98% or99% of the nucleotide bases, for example, 85%, 90%, 95%, 96%, 98%, or99% sequence identity as measured by any known algorithm of sequenceidentity, such as FASTA, BLAST or Gap.

As applied to polypeptides, the term “substantial identity” or“substantial similarity” means that two amino acid sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weight as supplied with the programs, share at least about 70%, 75%,80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity; e.g., 70%, 75%,80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. In certainembodiments, residue positions that are not identical differ byconservative amino acid substitutions.

“Therapeutically effective amount” refers to that amount of atherapeutic agent being administered that will ameliorate at least onesign or symptoms a disease being treated or enhance or improve theprophylactic and or therapeutic effect(s) of another therapy (e.g.,another therapeutic agent) useful for treating an IL-6 associateddisease. It is understood that the therapeutically effective amount maybe administered in multiple doses over a limited amount of time or as achronic treatment.

“Treat”, “treating” and “treatment” refer to a method of ameliorating asigns or symptoms or a disease.

As used herein, the term “disease” includes diseases and disorders.

The entire disclosure of each patent document and scientific articlereferred to herein, and those patent documents and scientific articlescited thereby, is expressly incorporated by reference herein for allpurposes.

Additional features and advantages of the invention are moreparticularly described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating results of an experiment in which ananti-IL-6 antibody was administered IVT in rat CNV model. Anti-VEGFantibody was administered as a positive control and the negative controlwas vehicle alone. p=0.0054 on Day 15 and p=0.0005 on Day 22 foranti-IL-6 vs. vehicle control.

FIG. 2 is a graph illustrating results of a binding experiment testingthe ability of the murine 64 antibody to inhibit binding of IL-6/IL-6Rto gp130.

FIG. 3A is a graph illustrating an experiment in which 020 was testedfor the ability to block IL-6 signaling in the absence of an excess ofsoluble IL-6Rα. Experiments were performed in HEK-Blue-IL-6 cells with0.2 ng/mL IL-6 and 2 μg/mL IL6Rα.

FIG. 3B is a graph illustrating an experiment in which 020 was testedfor the ability to block IL-6 signaling in the absence presence of anexcess of soluble IL-6Rα. Experiments were performed in HEK-Blue-IL-6cells with 0.2 ng/mL IL-6 and 2 μg/mL IL6Rα.

FIG. 4 is a graph illustrating the results of an experiment in which amonoclonal anti-IL-6 antibody (“IL-6 Blockade”) was administered IVT ina mouse CNV model. Controls were no treatment (contralateral eye),intravitreal injection of an anti-VEGF antibody (“VEGF Blockade”) orintravitreal injection of an anti-HRP isotype control antibody (“ControlAntibody”).

DETAILED DESCRIPTION

IL-6 has been implicated as playing a role in a number of diseases suchas rheumatoid arthritis, and has been reported to be significantlyup-regulated in a number of diseases, including ocular diseases. IL-6can act via both cis- and trans-mechanisms. In the cis mechanism, it isbelieved that free IL-6 binds to membrane bound IL-6 receptor (IL-6R isalso referred to as IL-6Rα and CD126), and the IL-6/IL-6R complex theninteracts with gp130 (also referred to as CD130, oncostatin M receptor,IL-6Rbeta, and IL-6 signal transducer), to activate signaling in thecell containing the complex. In the trans mechanism, free IL-6 binds tosoluble IL-6 receptor (sIL-6R). The IL-6/sIL-6R complex can then bind togp130 present in a cell membrane. A key difference between thesemechanisms is that more cell types express gp130 than express IL-6R,whose expression is more limited. Therefore, in diseases for which it isdesirable to inhibit IL-6 signaling, for example in those in which it isdesirable to broadly inhibit IL-6 signaling, it is useful to inhibitboth cis- and trans-IL-6 signaling. Applicants have engineered IL-6antagonists, e.g., anti-IL-6 antibodies, fragments, and derivatives thatcan inhibit both cis and trans signaling by IL-6. In addition,applicants have engineered such IL-6 antagonists to achieve enhancedvitreal retention and more rapid systemic clearance.

Features of IL-6 Antagonists (IL-6a)

In general, an IL-6 antagonist (IL-6a) described herein specificallybinds to site II (site 2) of an IL-6 and is useful for treatment of IL-6related eye disease and certain other diseases. An IL-6 related eyedisease is one in which an undesirable symptom or biological activity ofthe disease is associated with the expression or presence of IL-6. Insome embodiments the IL-6a has high affinity for both free and boundIL-6, is relatively stable in an organism, can inhibit binding to gp130of an IL-6 bound to an IL-6R (termed herein an IL-6/IL-6R complex orIL-6/IL-6R), and can have a therapeutic effect. In general, the IL-6a isan antibody or is derived from an antibody. For example, an IL-6a is ahigh affinity, humanized Fab that can specifically bind to site II of anIL-6 and potently blocks both cis- and trans-IL-6 signaling. In anotherexample, the IL-6a is a full length antibody, e.g., an IgG1 or IgG2antibody.

In some embodiments, the Fab is also configured as an Fc-engineeredsequence or is in a full-length antibody. In some embodiments, theFc-engineered IL-6a (e.g., the Fc-engineered Fab) has increased vitrealresidence time and/or more rapid systemic clearance compared with anappropriate control, e.g., compared with the corresponding antibody,fragment, or derivative thereof that does not have the engineered Fc.These and other features of an IL-6a are further described herein.

Applicants have designed IL-6 antagonists that selectively bind to siteII of IL-6 to provide broad inhibition of IL-6 signaling because suchmolecules can inhibit the binding of gp130 to IL-6, regardless ofwhether the IL-6 is bound to membrane IL-6R or sIL-6R. Furthermore,targeting the ligand (IL-6) as opposed to the IL-6 receptor can avoidreceptor mediated clearance and toxicity due to ADCC (antibody-dependentcell-mediated cytotoxicity). Because IL-6 plays both pathologic anddprotective roles in disease, use of an IL-6 antagonist (IL-6a) to treata disease associated with increased IL-6 can improve certain aspects ofa condition, but may also cause significant adverse effects, e.g.,systemic effects. This duality of IL-6 pathways (i.e., the ability tohave desirable and/or undesirable effects) can make it undersirable totreat an IL-6 associated disorder with a systemic inhibitor.Accordingly, the compositions and methods provided herein can be usefulfor treatments that inhibit at least one IL-6 activity, but do not havean undue effect on positive activities of IL-6, in part because thecompositions can be formulated for local delivery, e.g., for localdelivery to the eye. For example, in certain aspects, the IL-6a isdesigned to be of a size suitable for delivery to a particular site. Insome embodiments, the IL-6a is a full-length antibody. In someembodiments, the IL-6a is derived from an antibody and is in a formatthat may have longer residency in the vitreous of the eye and limitedsystemic leakage. In some embodiments, the IL-6a is a modified antibody(e.g., an antibody with a modified Fc domain) that has longer residencyin the vitreous of the eye and/or more limited systemic leakage comparedwith a corresponding unmodified antibody. In some embodiments, the IL-6ais an IgG2 antibody.

In some aspects, the IL-6a is a relatively small IL-6a such as afragment of an antibody or other derivative of an antibody that is lessthan a full length antibody, e.g., a Fab that is derived from an IL-6antibody. In some cases, an IL-6a is in a format that can pass from onepart of a tissue to another with increased kinetics compared to acorresponding full-length IL-6 antibody. In some embodiments, the IL-6ais a Fab that has been engineered to be a larger molecule, which is morelikely to have increased residence in the location to which it wasdelivered compared to the Fab alone, e.g., the IL-6a is dimerizedthrough Fc domain. In certain embodiments, the Fc domain has beenengineered such that the Fc moiety has ablated or reduced FcRn bindingthat can result in extended local residence, e.g., increased vitrealretention and reduce systemic accumulation compared to the same IL-6binding entity that includes a wild-type Fc.

The IL-6 antagonists described herein also have a sufficiently highaffinity for their target, IL-6 to be effective in ameliorating at leastone undesirable effect of IL-6. The inhibitors are also sufficientlystable to be useful as therapeutics.

In general, the PK of an IL-6a, e.g., an IL-6a suitable for use in theeye has a PK in the site of delivery, e.g., the vitreous, that issufficient to provide a therapeutic effect. In non-limiting examples,the PK can be a half-life of at least 12 hours, 24 hours, 2 days, 3days, 4 days, 5 days, 8 days, 10 days, 14 days, 21 days, 28 days, or 30days.

Identification of IL-6 Antagonists Binding to Site II

In general, any method known in the art can be used to generate amolecule that can bind to an IL-6, for example, polypeptide libraries ormolecular libraries can be screened for candidate compounds in an assayfor the ability of a polypeptide or compound to bind to IL-6. Once sucha candidate compound is identified, the binding site of the compound canbe determined using methods known in the art. For example, a moleculecan be tested for the ability to bind to wild type IL-6 and the bindingcompared to the ability of the compound to bind to an IL-6 mutated insite I, site II, or site III. In embodiments, an IL-6a as describedherein retains the ability to bind to an IL-6/IL-6Rα complex and toIL-6, and prevents binding of IL-6/IL-6Rα to gp130. In embodiments, anIL-6a as described herein can compete with gp130 for binding toIL-6/IL-6Rα complex, e.g., by binding to site II of IL-6. Such bindingactivities can be assayed using methods known in the art.

IL-6a candidates can be tested, for example, using an HEK-Blue™ IL-6assay system (InvivoGen, San Diego). HEK-Blue™ IL-6 cells are HEK293cells that are stably transfected with human IL-6R and a STAT3-inducibleSEAP reporter gene. In the presence of IL-6. STAT3 is activated and SEAPis secreted. SEAP is assessed using, for example, QUANT1-Blue™(InvivoGen, San Diego). Addition of an IL-6 antagonist to the cellsprevents secretion or decreases the level of SEAP as a result ofinhibiting both free and soluble receptor bound IL-6.

K_(D) refers to the binding affinity equilibrium constant of aparticular antibody-antigen interaction or antibody fragment-antigeninteraction. An antibody or fragment thereof is said to specificallybind an antigen when the K_(D) is less than or equal to 250 pM, e.g.,less than or equal to 225 pM, 220 pM, 210 pM, 205 pM, 150 pM, 100 pM, 50pM, 20 pM, 10 pM, or 1 pM, K_(D) can be determined using methods knownin the art, for example using surface plasmon resonance, for example,using the BiaCore™ system.

K_(off) refers to the dissociation rate constant of a particularantibody-antigen interaction or antibody fragment-antigen complex. Thedissociation rate constant can be determined using surface plasmonresonance, for example using the BiaCore™ system. A relatively slowK_(off) can contribute to desirable features of a therapeutic, e.g.,permitting less frequent administration of the inhibitor to a subject inneed of such treatment.

Specificity

A feature of IL-6 antagonists disclosed herein relates to their bindingspecificity. As discussed supra, IL-6 can be present as free IL-6 and asIL-6 bound to soluble IL-6Rα. Applicants have identified site II or IL-6as an optimal target for an IL-6 antagonist compared to an inhibitorthat that binds to site I of an IL-6. A site I inhibitor may inhibitbinding of free IL-6 to IL-6Rα. However, such an inhibitor cannotprevent activity initiated by pre-existing IL-6/IL-6R complexes exceptby replacement limited by the K_(off) of the complex. Anotheralternative, an inhibitor that binds to an IL-6Rα, is less suitablebecause it may have limited ability to prevent IL-6 activity unless itis present in saturating concentrations. Because the amount of IL-6receptor is generally quite high compared to the amount of IL-6, thisapproach may require the administration of an undesirably large amountof a composition that inhibits IL-6 activity by binding to the receptor.In embodiments, the IL-6 antagonists described herein (e.g., theantibodies and fragments and derivatives thereof described herein) canblock the activity of IL-6 even when IL-6 is bound to IL-6R.Accordingly, an advantage of an IL-6a as described herein is thatrelatively less of the composition may need to be administered toachieve a therapeutic effect compared to an inhibitor targeting an IL-6receptor. Anti-receptor antibodies have been reported to be clearedrapidly by receptor mediated clearance significantly limiting their PK,therefore requiring larger doses, more frequent dosing, or both.Additionally, both anti-receptor and anti-site I IL-6 antibodies pose aproblem in that they significantly increase the tissue concentration ofIL-6 by disrupting the normal receptor mediated clearance pathway of theligand, thereby exposing the subject to potentially undesirable levelsof IL-6 in a tissue. Furthermore, use of an inhibitor targeting IL-6Rαmay necessitate the presence of the inhibitor near both sites at whichinhibition is sought and a site at which it is not desirable, e.g.,systemic treatment. Use of an IL-6a that binds site II, the site towhich gp130 binds, permits inhibition via free IL-6 as well as IL-6 thatis bound to an IL-6R, but has not yet activated an IL-6 pathway viagp130. Accordingly, the IL-6 antagonists described herein are designedto bind to both forms of IL-6 (soluble and receptor bound), specificallythe IL-6 antagonists bind to site II of IL-6, which is accessible inboth forms. Compositions containing an IL-6a as described herein caninhibit both cis and trans signaling by IL-6.

In some cases compounds and methods provided herein are designed toprovide an effective IL-6 blockade sufficient to treat at least one signor symptom of an IL-6 associated disorder, for example, inhibitingangiogenesis and/or inflammation.

Compounds described herein are useful for treating eye diseasescharacterized by an undesirably high level of IL-6, e.g., in thevitreous (see Yuuki et al., J Diabetes Compl 15:257 (2001); Funatsu etal., Ophthalmology 110:1690, (2003); Oh et al., Curr Eye Res 35:1116(2010); Noma et al., Eye 22:42 (2008); Kawashima et al., Jpn JOphthalmol 51:100 (2007); Kauffman et al., Invest Ophthalmol Vis Sci35:900 (1994); Miao et al., Molec Vis 18:574(2012)).

In general, an IL-6a as described herein is a potent antagonist of IL-6signaling. This is achieved in part by designing molecules having a highaffinity for IL-6, for example, an IC50 less than or equal to 100 pM inan HEK-Blue IL-6 assay using 10 pM IL-6. High affinity of an IL-6a canbe determined based on the K_(D) of the IL-6a, for example, a K_(D) ofless than or equal to 1 nM, less than or equal to 500 pM, less than orequal to 400 pM, less than or equal to 300 pM, less than or equal to 240pM, or less than or equal to 200 pM.

To produce a biologic IL-6a (e.g., a protein or polypeptide such as anantibody, fragment, or derivative thereof) that is useful for treating adisorder associated with increased IL-6 expression or activity,typically it is desirable that the biologic IL-6a have highproductivity. For example, a suitable productivity is greater than orequal to 1 g/L (e.g., greater than or equal to 2 g/L, greater than orequal to 5 g/L, or greater than or equal to 10 g/L).

To effectively administer an IL-6 antagonist, it is necessary that theinhibitor have solubility compatible with the concentration at which itwill be administered. For example, in the case of a full-length antibodyIL-6a, the solubility is greater than or equal to 20 mg/ml, greater thanor equal to 10 mg/ml, greater than or equal to 5 mg/ml, or greater thanor equal to 1 mg/ml.

Furthermore, to be a viable treatment, the inhibitor must have highstability at the body temperature of the delivery and activity sites aswell as storage stability. For example, a T_(ro) of greater than orequal to 60°C. (e.g., greater than or equal to 60°C., greater than orequal to 62.5° C., greater than or equal to 65° C., greater than orequal to 70° C., greater than or equal to 73° C., greater than or equalto 75° C.) and a T_(onset) of greater than or equal to 45° C., e.g.,greater than or equal to 50° C., greater than or equal to 51° C.,greater than or equal to 55° C., or greater than or equal to 60° C.Methods of determining the T_(m) and T_(onset) can be determined usingmethods known in the art.

Antagonists having the desired features can be selected from suitabletypes of molecules known in the art, for example antibodies, includingfragments and derivatives of an IL-6 site II targeted antibody thatgenerally retains or maintains sufficient features of the parent IL-6antibody (e.g., desired binding properties). Such antagonists includeF_(ab) fragments, scFvs, F_(ab) fragments engineered to include an Fcmoiety, and full-length antibodies engineered to have a frameworkdifferent from the parent IL-6 site II targeted antibody.

In some aspects, the IL-6a disclosed herein comprises a human antibodyantigen-binding site that can compete or cross-compete with an antibodyor fragment thereof that can bind to site II of IL-6. For example, theantibody or fragment thereof can be composed of a VH domain and a VLdomain disclosed herein, and the VH and VL domains comprise a set ofCDRs of an IL-6/site II binding antibody disclosed herein.

Any sutable method may be used to determine the domain and/or epitopebound by an IL-6a, for example, by mutating various sites on an IL-6.Those sites in which mutations prevent or decrease binding of the IL-6aand the IL-6 ligand are involved either directly in binding to the IL-6aor indirectly affect the binding site, e.g., by affecting conformationof the IL-6. Other methods can be used to determine the amino acidsbound by an IL-6a. For example, a paptide-binding scan can be used, suchas a PEPSCAN-based enzyme linked immuno assay (ELISA). In apeptide-binding scan of this type, short oerlapping peptides derivedfrom the antigen are systematically screened for binding to a bindingmember. The peptides can be covalently coupled to a support surface toform an array of peptides. Peptides can be in a linear or constrainedconformation. A constrained conformation can be produced using peptideshaving a terminal cysteine (cys) residue at each end of the peptidesequence. The cys residues can be covalently coupled directly orindirectly to a support surface such that the peptide is held in alooped conformation. Accordingly, a peptide used in the method may havea cys residue added to each end of a peptide sequence corresponding to afragment of the antigen. Double looped peptides can also be used, inwhich a cys residue is additionally located at or near the middle of thepeptide sequence. The cys residues can be covalently coupled directly orindirectly to a support surface such that the peptides form adouble-looped conformation, with one loop on each side of the centralcys residue. Peptides can by synthetically generated, and cys residuescan therefore by engineered at desired locations, despite not occurringnaturally in the IL-6 site II sequence. Optionally, linear andconstrained peptides can both be screened in a peptide-binding assay. Apeptide-binding scan may involve identifying (e.g., using an ELISA) aset of peptides to which the binding member binds, wherein the peptideshave amino acid sequences corresponding to fragments of an IL-6a (e.g.,peptides that include about 5, 10, or 15 contiguous residues of anIL-6a), and aligning the peptides in order to determine a footprint ofresidues bound by the binding member, where the footprint comprisesresidues common to overlapping peptides. Alternatively or additionallythe peptide-binding scan method can be used to identify peptides towhich the IL-6a binds with at least a selected signal:noise ratio.

Other methods known in the art can be used to determine the residuesbound by an antibody, and/or to confirm peptide-binding scan results,including for example, site directed mutagenesis (e.g., as describedherein), hydrogen deuterium exchange, mass spectrometry, NMR, and X-raycrystallography.

Typically, an IL-6a useful as described herein is a human antibodymolecule, a humanized antibody molecule, or binding fragment thereof. Ingeneral, the antibody is a monoclonal antibody. The origin of such anantibody can be human, murine, rat, camelid, rabbit, ovine, porcine, orbovine and can be generated according to methods known to those in theart.

In general, an IL-6a comprises at least the CDRs of an antibody that canspecifically bind to site II of an IL-6 (e.g., a human IL-6). Thestructure for carying a CDR or a set of CDRs of the invention can be anantibody heavy or light chain sequence or substantial portion thereof inwhich the CDR or set of CDRs is located at a location corresponding tothe CDR or set of CDRs of naturally occurring VH and VL antibodyvariable domains encoded by rearranged immunoglobulin genes. Thestructures and locations of immunoglobulin variable domains can bedetermined by reference to Kabat, et. al., 1983 (National Institutes ofHealth), and updates thereof findable under “Kabat” using any internetsearch engine.

An IL-6a that is an antibody generally comprises an antibody VH domain(e.g., SEQ ID NO:1 or SEQ ID NO:3) and/or VL domain (e.g., SEQ ID NO:2).A VH domain comprises a set of heavy chain CDRs (VHCDRs), and a VLdomain comprises a set of light chain CDRs (VLCDRs). Examples of suchCDRS are provided in Example 3, examples of which are illustrated in SEQID NOs:4-9. An antibody molecule can comprise an antibody VH domaincomprising a VHCDR1, VHCDR2 and VHCDR3 and a framework. It canalternatively or also comprise an antibody VL domain comprising aVLCDR1, VLCDR2 and VLCDR3 and a framework.

Disclosed herein are IL-6 antagonists comprising a VHCDR1 and/or VHCDR2and/or VHCDR3 such as those disclosed herein and/or a VLCDR1 and/orVLCDR2 and/or VLCDR3 such as those disclosed herein. The IL-6a cancomprise one or more CDRs of any of the antibodies, fragments orderivatives described herein. The IL-6a can comprise a set of VHCDRs(e.g., VHCDR1, VHCDR2, and VHCDR3), and optionally it can also comprisea set of VLCDRs (e.g., VLCDR1, VLCDR2, and VLCDR3). The CDRs can bederived from one or more antibodies, fragments, or derivatives describedherein. For example, the VLCDRs can be derived from the same or adifferent antibody as the VHCDRs.

In general, a VH domain is paired with a VL domain to provide anantibody antigen-binding site. For example, the HC domain of SEQ ID NO:1or SEQ ID NO:3 is paired with the LC domain of SEQ ID NO:2. In somecases, a VH or VL domain alone can be used as an IL-6a.

In some aspects, the IL-6a is an antibody molecule, fragment, orderivative thereof that comprises (i) a VH domain sequence that has atleast 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identitywith a VH domain described herein, e.g., a VH domain of SEQ ID NO:1 orSEQ ID NO:3, or (ii) a set of VHCDRs (e.g., VHCDR1, VHCDR2, and/orVHCDR3) of those sequences (the sequences defined in (i)). Inembodiments, the antibody molecule, fragment, or derivative thereofcomprises a VHCDR1, VHCDR2, and VHCDR3 of SEQ ID NO:1 or a VHCDR1,VHCDR2, and VHCDR3 of SEQ ID NO:3. In embodiments, the antibodymolecule, fragment, or derivative thereof comprises a VHCDR1, VHCDR2,and VHCDR3 that collectively differ from the VHCDR1, VHCDR2, and VHCDR3of SEQ ID NO:1 by no more than 1, no more than 2, no more than 3, nomore than 4, or no more than 5 amino acids. In embodiments, the antibodymolecule, fragment, or derivative thereof comprises a VHCDR1, VHCDR2,and VHCDR3 that collectively differ from the VHCDR1, VHCDR2, and VHCDR3of SEQ ID NO:3 by no more than 1, no more than 2, no more than 3, nomore than 4, or no more than 5 amino acids;

The antibody molecule, fragment, or derivative thereof can optionallyalso comprise (i) a VL domain sequence that has at least 60, 70, 80, 85,90, 95, 98 or 99% amino acid sequence identity with a VL domaindescribed herein, e.g., a VL domain of SEQ ID NO:2, or (ii) a set ofVLCDRs (e.g., VLCDR1, VLCDR2, and/or VLCDR3) of those sequences (thesequences defined in (i)). In embodiments, the antibody molecule,fragment or derivative thereof comprises VLCDR1, VLCDR2, and VLCDR3 ofSEQ ID NO:2. In embodiments, the antibody molecule, fragment, orderivative comprises a VLCDR1, VLCDR2, and VLCDR3 that collectivelydiffer from the VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NO:3 by no morethan 1, no more than 2, no more than 3, no more than 4, or no more than5 amino acids. Algorithms that can be used to calculate percent identityof two amino acid sequences include e.g., BLAST, FASTA, or theSmith-Waterman algorithm, e.g., employing default parameters.

An IL-6a as described herein can comprise antibody constant regions orparts thereof, e.g., human antibody constant regions or parts thereof.For example, a VL domain may be attached at its C-terminal end toantibody light chain constant domains including human CK or CL chains.Similarly, and IL-6a based on a VH domain can be attached at itsC-terminal end to all or part (e.g., a CH1 domain) of an immunoglobulinheavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE andIgM and any of the isotype sub-classes, particularly IgG1, IgG2, IgG3and IgG4.

In some cases, an antibody of the invention is further modified usingmethods known in the art create a sequence having a specific allotype,for example an allotype that predominates in a population having aparticular geographic origin. In some cases, the human heavy chainconstant region is modified for this purpose.

An IL-6a can be an antibody molecule, binding fragment thereof, orvariant, having one or more CDRs, for example, a set of CDRs, within anantibody framework. For example, one or more CDRs or a set of CDRs of anantibody (e.g., an antibody or frament or derivative thereof asdescribed herein) may be grafted into a framework (e.g., humanframework) to provide an antibody molecule. The framework regions can bederived from human germline gene sequences, or be non-germline inorigin.

VH and/or VL framework residues can be modified as discussed andexemplified herein e.g., using site-directed mutagenesis.

Amino acid changes can be made in one or more framework regions and/orone or more CDRs derived from an antibody IL-6a targeted to site II ofIL-6 (termed herein a “reference IL-6 antibody”) using methods andparameters known in the art. Also included herein is a resulting IL-6antagonist that retains binding to site II of an IL-6 (e.g., site II ofa human IL-6) and typically has at least the same binding or increasedaffinity compared to the reference IL-6 antibody. In some cases, toimprove a parameter such as stability, a change that results in adecrease in binding affinity of the derived IL-6a compared to thereference IL-6a (e.g., the reference antibody) can be introduced tocreate a useful IL-6a. In some embodiments, e.g., in some cases in whichthe reference relates to FcRn binding or a pharmacokinetic (PH)parameter such as half-life in the vitreous or systemic half-life (e.g.,in blood, plasma, serum, lymph, liver, kidney, other tissue, or bodyfluid), a reference antibody may be an antibody that does notspecifically bind an IL-6.

A change in the amino acid sequence of an IL-6a polypeptide can includesubstituting one or more amino acid residue(s) with a non-naturallyoccurring or non-standard amino acid, modifying one or more amino acidresidue into a non-naturally occurring or non-standard form, orinserting one or more non-naturally occurring or non-standard amino acidinto the sequence. Examples of numbers and locations of alterations insequences of the invention are described elsewhere herein. naturallyoccurring amino acids include the 20 “standart” L-amino acids identifiedas G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E by theirstandard single-letter codes. Non-standard amino acids include any otherresidue that may be incorporated into a polypeptide backbone or resultfrom modification of an existing amino acid residue. Non-standard aminoacids may be naturally occurring or non-naturally occurring. Severalnaturally occurring non-standard amino acids are known in the art, suchas 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, andN-acetylserine. Those amino acid residues that are derivatized at theirN-alpha position will only be located at the N-terminus of an amino-acidsequence. The amino acid is typically an L-amino acid. In some cases theamino acid is a D-amino acid. Alteration may therefore comprisemodifying an L-amino acid into, or replacing it with, a D-amino acid.Methylated, acetylated and/or phosphorylated forms of amino acids arealso known, and amino acids in the present invention may be subject tosuch modification.

Amino acid sequences in antibody domains and binding members of theinvention can comprise non-natural or non-standard amino acids asdiscussed herein. Non-standard amino acids (e.g., D-amino acids) can beincorporated into an amino acid sequence using methods known in the art,for example in synthesis of the molecule or by post-synthesismodification or replacement of an amino acid. In some cases, a D-aminoacid is used to increase PK of an IL-6a.

Novel VH or VL regions carrying CDR-derived sequences of the inventionmay be generated using random mutagenesis of one or more selected VHand/or VL nucleic acid sequences to generate mutations within the entirevariable domain. For eample, error-prone PCR can be used (Chao et al.,Nature Protocols, 1:755-768 (2006)). In some embodiments one or twoamino acid substitutions are made within an entire variable domain orset of CDRs. Other methods know in the art can be used to generatemutations, for example site-directed mutagenesis, typically in one ormore CDRs.

One method for producing an antibody IL-6a, is to alter a VH domain suchas those depicted in SEQ ID NO:1 and SEQ ID NO:3 by adding, deleting,substituting or inserting one or more amino acids. The altered VH domaincan be combined with a VL domain such as that depicted in SEQ ID NO:2,which can also be altered as described herein and using methods known inthe art. Such altered molecules are tested for their ability to bind tosite II of IL-6 and optionally for other desired properties such asincreased affinity compared to a reference molecule. In some cases, avariant VH or VL domain can have 1, 2, 3, 4, or 5 such alterations(e.g., 1, 2, 3, 4, or 5 amino acid substitutions).

An IL-6a of the invention can be a fragment of an antibody that binds tosite II of an IL-6 provided that the fragment comprises an antigenbinding site, e.g., can bind to site II of an IL-6. Antibody fragmentsof the invention are generally obtained starting with a reference(parent) antibody molecule such as an antibody molecule comprising SEQID NO:1 and SEQ ID NO:2 or SEQ ID NO:3 and SEQ ID NO:2. Antibodyfragments can be generated using methods known in the art such asrecombinant DNA, enzymatic cleavage (for example, using pepsin orpapain), chemical cleavage of an antibody (for example, chemicalreduction of disulfide bridges). Antibody fragments that comprise anantibody antigen-binding site include, but are not limited to, moleculessuch as Fab, Fab′, Fab′-SH, scFv, Fv, dAb, Fd, and disulfide stabilizedvariable region (dsFv). Various other antibody molecules including oneor more antibody antigen-binding sites can be engineered, including forexample F(ab′)2, F(ab)3, diabodies. triabodies, tetrabodies, andminibodies. Examples of antibody molecules and methods for theirconstruction and use are described in Holliger and Hudson, 2005, NatBiotechnol 23:1126-1136. Non-limiting examples of binding fragments area Fab fragment composed of VL, VH, constant light chain domain (CL) andconstant heavy chain domain 1 (CH1) domains; an Fd fragment composed ofVH and CH1 domains; an Fv fragment composed of the VL and VH domains ofa single antibody; a dAb fragment composed of a VH or a VL domain;isolated CDR regions; an F(ab′)2 fragment, a bivalent fragmentcomprising two linked Fab fragments; a single chain Fv molecule (scFv),in which a VH domain and a VL domain are linked by a paptide linkerwhich allows the two domains to associate to form an antigen bindingsite; a bispecific single chain Fv dimer (for example as disclosed in WO1993/011161) and a diabody, which is a multivalent or multispecificfragment constructed using gene fusion (for example as disclosed inWO94/13804), Fv, scFv, or diabody molecules can be stabilized by theincorporation of disulfide bridges linking the VH and VL domains.Minibodies comprising an scFv joined to a CH3 domain can also be used asan IL-6a. Other fragments and derivatives of an antibody that can beused as an IL-6a include a Fab′, which differs from a Fab fragment bythe addition of a few amino acid residues at the carbodyl terminus ofthe heavy chain CH1 domain, including one or more cysteines from theantibody hinge region, and Fab′-SH, which is a Fab′ fragment in whichthe cysteine residue(s) of the constant domains bear a free thiol group.

In some cases, an IL-6a that is an antibody fragment has been chemicallymodified to improve or introduce a desirable property, for examplePEGylation to increase half-life or incorporation.

A dab (domain antibody) is a small monomeric antigen-binding fragment ofan antibody (the variable region of an antibody heavy or light chain. VHdAbs occur naturally in camelids (e.g., camels and Hamas) and can beproduced by immunizing a camelid with a target antigen, isolatingantigen-specific B cells and directly cloning dAb genes from individualB cells. An IL-6a of the present invention can be a dAb comprising a VHor VL domain substantially as set out herein, or a VH or VL domaincomprising a set of CDRs substantially as set out herein.

Antibodies of the invention include bispecific antibodies in which twodifferent variable regions are combined in the same molecule. An IL-6acan be incorporated as part of a bispecific antibody prepared usingmethods known in the art, for example, prepared chemically or fromhybrid hybridomas. Such a molecule can be a bispecific antibody fragmentof a type discussed above. One non-limiting example of a method forgenerating a bispecific antibody is BiTE™ technology in which thebinding domains of two antibodies with different specificity can be usedand directly linked via short flexible peptides. this combines twoantibodies on a short single polypeptide chain. Diabodies and scFv canbe constructed without an Fc region, using only variable domains,potentially reducing the effects of anti-idiotypic reaction. Bispecificantibodies can be constructed as entire IgG, as bispecific Fab′2, asFab′PEG, as diabodies or else as bispecific scFv. Further, twobispecific antibodies can be linked using routine methods known in theart to form tetravalent antibodies.

Bispecific diabodies, as opposed to bispecific whole antibodies, areuseful, in part because they can be constructed and expressed in E.Coli. Diabodies (and many other polypeptides, such as antibodyfragments) of appropriate binding specificities can be readily selectedusing phage display (WO 1994/13804) from libraries. If one arm of thediabody is to be kept constant, for example, with a specificity directedagainst site II of IL-6, then a library can be made where the other armis varied and an antibody of appropriate specificity selected.

Bispecific whole antibodies may be made by alternative engineeringmethods as described in described in WO 1996/27011, WO 1998/50431 and WO2006/028936.

In some cases, an IL-6a of the invention comprises an antigen-bindingsite within a non-antibody molecule, for example, by incorporating oneor more CDRs e.g. a set of CDRs in a non-antibody protein scaffold, asdiscussed further below. In some cases, the CDRs are incorporated into anon-antibody scaffold. An IL-6 site II binding site can be provided byan arrangement of CDRs on non-antibody protein scaffolds, such asfibronectin or cytochrome B, or by randomizing or mutating amino acidresidues of a loop within a protein scaffold to confer bindingspecificity for an IL-6 site II. Scaffolds for engineering novel bindingsites in proteins are known in the art. For example, protein scaffoldsfor antibody mimics are disclosed in WO200034784, which describesproteins (antibody mimics) that include a fibronectin type III domainhaving at least one randomized loop. A suitable scaffold into which tograft one or more CDRs, e.g., a set of HCDRs, can be provided by anydomain member of the immunoglobulin gene superfamily. The scaffold canbe a human or non-human protein. An advantage of a non-antibody proteinscaffold is that it can provide an antigen-binding site in a scaffoldmolecule that is smaller and/or easier to manufacture than at least someantibody molecules. Small size of a binding member may confer usefulphysiological properties, such as an ability to center cells, penetratedeep into tissues or reach targets within other structures, or to bindwithin protein cavities of the target antigen. Typical are proteinshaving a stable backbone and one or more variable loops, in which theamino acid sequence of the loop or loops is specifically or randomlymutated to create an antigen-binding site that binds the target antigen.Such proteins include the IgG-binding domains of protein A from S.aureus, transferrin, tetranectin, fibronectin (e.g., using the 10thfibronectin type III domain), lipocalins as well as gamma-crystallineand other Affilin™ scaffolds (Scil Proteins, Halle, Germany). Examplesof other approaches include synthetic microbodies based oncyclotides—small proteins having intra-molecular disulfide bonds,microproteins (e.g., Versabodies™, Amunix Inc., Mountain View, Calif.)and ankyrin repeat proteins (DARPins, e.g., from Molecular Partners AG,Zurich-Schlieren, Switzerland). Such proteins also include small,engineered protein domains such as, for example, immuno-domains (see forexample, U.S. Patent Publication Nos. 2003/082630 and 2003/157561).Immuno-domains contain at least one complementarity determining region(CDR) of an antibody.

An IL-6a can comprise additional amino acids, e.g., to impart to themolecule another functional characteristic in addition to ability tobind antigen.

In some cases, an IL-6a carries a detectable label, or is conjugated toa toxin or a targeting moiety or enzyme (e.g., via a paptidyl bond orlinker). For example, an IL-6a can comprise a catalytic site (e.g., inan enzyme domain) as well as an antigen binding site (e.g., binding sitefor site II of an IL-6), such that the antigen binding site binds to theantigen and thus targets the catalytic site to IL-6 or IL-6/IL-6Rcomplex. The catalytic site can, in some cases, further inhibit abiological function of an IL-6, e.g., by cleavage of the IL-6, IL-6R, orother molecule that is associated with the IL-6a/IL-6 complex.

In some aspects, the invention includes an antibody IL-6a that has beenmodified compared to a regerence antibody to alter, for example,increase, decrease, or eliminate, the biological effect function of theIL-6a. In one example, the Fc region is modified or the parental Fcdomain is replaced with a modified Fc domain to alter thepharmacokinetics of the modified IL-6a compared to the unmodifiedparent. In some embodiments, the IL-6a is engineered to have an IgG2framework. In other embodiments, the IL-6a in an IgG1 or IgG2 frameworkhas a modified Fc that increases the binding affinity of the IL-6a at pH6.0 and does not substantially alter the binding affinity at pH 7.0compared to a parent or other reference IL-6a, or the Fc domain ismodified and the IL-6a has an increased half-life compared to a parentor other reference IL-6a.

In some embodiments, an antibody IL-6a is modified to increasecomplement fixation and complement-dependent cytotoxicity. In otheraspects, the antibody IL-6a is modified to increase the ability of theantibody compared to a reference antibody to activate effector cells andparticipate in antibody-dependent cytotocicity (ADCC). In some cases,the antibodies as disclosed herein can be modified both to enhance theircapability of activating effector cells and participating inantibody-dependent cytotoxicity (ADCC) and to enhance their capabilityof fixing complement and participating in complement-dependentcytotoxicity (CDC).

In some embodiments, the antibodies disclosed herein are modified toreduce their ability to fix complement and participate incomplement-dependent cytotoxicity (CDC). In other embodiments, theantibodies are modified to reduce their ability to activate effectorcells and participate in antibody-dependent cytotoxicity (ADCC). In yetother embodiments, an antibody as disclosed herein can be modified bothto reduce its ability to activate effector cells and participate inantibody-dependent cytotoxicity (ADCC) and to reduce its ability to fixcomplement and participate in complement-dependent cytotoixcity (CDC).

It is generally advantageous to avoid frequent delivery of a dose of anIL-6a, for example, when delivered by injection into the eye. Tofacilitate this feature, in certain embodiments, the half-life at thesite of delivery, e.g., the vitreous, of an IL-6a as disclosed herein isat least 4 days, for example, at least 7 days, at least 9 days, at least11 days, or at least 14 days. In certain embodiments, the mean half-lifeof an IL-6a is at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days,8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16days, 17 days, 18 days, 19 days, 20 days, 25 days, 30 days, 40 days, 50days, or 60 days. Methods of increasing the half-life of an antibody areknown in the art, for example as described in U.S. Pat. No. 6,277,375and International Publication Nos. WO 1998/23289 and WO 1997/3461. Insome embodiments, the half-life of an IL-6a is greater at the targetdelivery site, e.g., the vitreous, than systemic half-life, e.g.,half-life in blood, serum, plasma, lymph, liver, kidney, or other tissueor body fluid).

In another embodiment, the invention provides an article of manufactureincluding a container. The container includes a composition containingan IL-6a as disclosed herein, and a package insert or label indicatingthat the composition can be used to treat an IL-6 related disorder.Typically, the composition is an IL-6a in a composition comprising apharmaceutically acceptable excipient.

In some cases, the invention is a kit comprising a compositioncontaining an IL-6a as disclosed herein, and instructions to administerthe composition to a subject in need of treatment.

In embodiments in which a large IL-6a is desirable, e.g., to enhanceretention of the IL-6a at or near its site of delivery, a moiety thatincreases size but does not significantly adversely affect function ofthe IL-6a (e.g., binding affinity of the IL-6 for IL-6 or IL-6/IL-6Rcomplex) can be associated with the IL-6a. For example, a Fab can begenetically engineered to be expressed as signle polypeptides containinga Fab and an Fc moiety.

In embodiments in which a relatively small size for the IL-6a isdesirable, fragments of an IL-6 antibody can be used, for example, asscFv or a Fab fragment. An IgG antibody is about 150 kD in size, a Fabis about 50 kD and an scFv is about 25 kD. In some embodiments, an IL-6aas described herein is less than about 50 kD in size. Such an antagonistcan be, for example, less than or equal to 50 kD and greater than 10 kD,less than or equal to 50 kD and greater than 20 kD, or less than orequal to 50 kD and greater than or equal to 25 kD.

In some cases, stability of an IL-6 antagonist, e.g., an antibody orother inhibitor having disulfides, is improved by creating variant inshich one or more of the disulfide bridges are more stable than in theparent molecule.

Another advantage of certain IL-6a molecules described herein can be theavailability of effective molecules having a size suitable for theirmode of delivery, site of delivery, or mode of activity. For example, anIL-6a in a Fab format may be used for a topical application. Methods ofengineering such molecules are described herein and are known in theart.

Indications/IL-6 Associated Disease

Diseases that can be treated with an IL-6a of the invention includethose diseases in which elevated IL-6 is associated with the diseasestate or as a prerequisite to the disease state. Such diseases includethose inwhich angiogenesis and inflammation driven by IL-6 contribute todisease pathology. This includes diseases in which IL-6 is elevatedcompared to normal levels, e.g., diseases in which IL-6 is eleated inthe vitreous (such as, e.g., diabetic macular edema, diabeticretinopathy, and uveitis) or tissues of the eye. Examples includecertain eye diseases including, without limitation, dry eye syndrome,uveitis, age-related macular degeneration (AMD), proliferative diabeticretinopathy (PDR), diabetic macular edema (DME), retinal vein occlusion(RVO), neuromyelitis optica (NMO). Other ocular disorders that can betreated include those caused by trauma such as corneal transplant,corneal abrasion, or other such physical injury to the eye. Accordingly,the invention includes treating a subject having an IL-6 related diseasewith an IL-6a described herein. In some embodiments, treatment of asubject also includes determining whether the subject has an IL-6associated disease, and optionally, whether the subject is resistant toother non-IL-6 inhibitory treatments such as steroids or anti-VEGFtherapeutics.

One problem with certain antibody-based therapeutics that are effectiveat a specific locus such as the eye, for example in the vitreous, isadverse effects that result from systemic administration. One solutionis to provide therapeutics that can be delivered locally as opposed tosystemically as exemplified by molecules described herein. Because sometherapeutics that are locally delivered, e.g., to the vitreous, will, tosome extent, appear systemically, it is advantageous to design amolecule that will have relatively rapid systemic turnover. Applicantshave engineered an example of such a molecule; an IL-6a antibody that isdesigned for rapid systemic turnover, e.g., compared to the parentalmolecule or a reference antibody. This was accomplished by modifyingFcRn binding of the molecule to reduce FcRn mediated recycling of theIL-6a. A further advantage of such engineering, i.e., reducing FcRnrecycling, can be an enhanced vitreal retention by reducing the effluxof antibodies from the vitreous following IVT administration.Accordingly, in some embodiments, an IL-6a having low FcRn binding candecrease dosing frequency.

Diabetic macular edema (DME). Diabetic macular edema (DME) involvesocclusion and leakage of retinal blood vessels, causing reduced visualacuity and potentially blindness. Standard treatments for DME includelocal administration of steroids or anti-VEGF antibodies. However, manypatients are refractory to these therapies. The pathogenesis of diabeticmacular edema involves components of angiogenesis, inflammation, andoxidative stress IL-6 is induced by hypozia and hyperglycemia and canincrease vascular inflammation, vascular permeability, and pathologicangiogenesis. IL-6 can directly induce VEGF expression and can promotechoroidal neovascularization in aminal models. In DME patients, ocularIL-6 levels are positively correlated with macular thickness and diseaseseverity. IL-6 levels are reportedly elevated in patients who failanti-VEGF therapy while decreasing in anti-VEGF responsive patients.Accordingly, administration of an IL-6a as described herein is usefulfor treatment of diabetics in combination with an anti-VEGF therapeuticor as an alternative to anti-VEGF treatment, including for patients, whodo not respond to anti-VEGF therapy. Treatment of macular edema with anIL-6a may also improve safety by removing the need to completely inhibiteither mechanism to inhibit the pathology, thus preserving some of thedesired, physiological roles of each cytokine. Accordingly, local IL-6atreatment in combination with VEGF inhibition can decrease the dosefrequency and reduce adverse effects of treatment.

In DME there are positive correlations between vitreal IL-6 levels andboth disease severity and VEGF refractory subjects. Accordingly, andIL-6a as described herein can be used to treat DME subjects who arerefractive to steroid therapy, anti-VEGF therapy, or both. In somecases, an IL-6a is used in combination with anti-VEGF therapy or steroidtherapy, e.g., to treat DME.

An IL-6a described herein can also be used to treat disorders such ascancer, e.g., prostate cancer, leukemia, multiple myeloma, inflammatory(such as chronic inflammatory proliferative diseases) and autoimmunedisease, e.g., rheumatoid arthritis. Castleman's disease (giant orangiofollicular lymph node hyperplasia, lymphoid hamartoma,angiofollicular lymph node hyperplasia), juvenile idiopathic arthrits(including polyarticular juvenile idiopathic arthritis and systemicjuvenile idiopathic arthritis). Still's disease (encompassing juvenileidiopathic arthritis and adult onset Still's disease), adult onsetStill'disease, amyloid A amylidosis, polymyalgia rheumatica, remittingseronegative symmetriacal synovitis with pitting edema,spondyloarthritides, Behcet's disease (including treatment of ocularmanifestations), atherosclerosis, psoriasis, systemic lupuserythematosis, polymyositis (an inflammatory myopathy), relapsingpolychondritis, acquired hemophilia A, multiple sclerosis, anemia ofinflammation, and Crohn's disease.

IL-6 antagonists are also useful for treatment of certain neurologicdiseases, for example, depression, and Alzheimer's disease.

Other diseases that can be treated with an IL-6a described hereininclude, without limitation, systemic sclerosis, Takayasu arteritis,giant cell arteritis, graft versus host disease, andTNF-receptor-associated periodic syndrome (TRAPS).

Dosing

An IL-6 antibody or fragment thereof can be administered to a subject(e.g., a patient) who expresses, e.g., abnormally high levels of IL-6.The antibody or fragment thereof can be administered once, or can beadministered multiple times. The antibody may be administered, forexample, from three times daily to once every six months or longer. Theadministration can be on a schedule such as three times daily, twicedaily, once daily, once every two days, once every three days, onceweekly, once every two weeks, once every month, once every two months,once every three months and once every six months. The antibody orfragment thereof can be administered continuously via a minipump orother route such as an implantable slow-release capsule or by anencapsulated cell producing the antibody or fragment thereof. Theantibody or fragment thereof can be administered via a mucosal, buccal,intranasal, inhalable, intravenous, subcutaneous, intramuscular,parenteral, intraocular, or intratumor route. The antibody or fragmentthereof can be administered once, at least twice or for at least theperiod of time until the condition is treated, palliated or cured. Theantibody or fragment thereof generally will be administered for as longas the condition is present. The antibody or fragment thereof, it willgenerally be administered as part of a pharmaceutical composition asdescribed herein. The dosage of antibody will generally be in the rangeof 0.1 to 100 mg/kg, 0.5 to 50 mg/kg, 1 to 20 mg/kg, and 1 to 10 mg/kg.The serum concentration of the antibody or fragment thereof can bemeasured by any suitable method. One feature of certain compoundsdescribed herein is that they require relatively infrequent dosing, forexample, once per week, twice per week, three times per week, once everyfour weeks, once every two weeks, once every 8 weeks, once every 12weeks, once every 16 weeks, once every 32 weeks, once per month, onceper two months, once per three months, or once per six months. In somecases the compound is administered on an as needed basis, determined,for example by a subject's condition. It is a feature of the IL-6antagonists described herein that permits relatively infrequent dosingis the combination of high potency which is accomplished, at least inpart, by a slow off rate once bound to an IL-6 and the ability todeliver a relatively high concentration of the compound.

In some cases, the IL-6a is administered as a monotherapy. In otherembodiments, the IL-6a is administered concomitantly with methotrexateor other disease modifying anti-arthritic drug.

Generation of Antibodies

An antibody IL-6a derivative or fragment thereof can be produced usingmethods known in the art such as monoclonal antibody methodology (e.g.,see Kohler and Milstein (1975) Nature 256:495). Other techniques forproducing monoclonal antibodies can also be employed such as viral oroncogenic transformation of B lymphocytes. Chimeric or humanizedantibodies can be prepared based on the sequence of a murine monoclonalantibody prepared using methods known in the art. DNA encoding the heavyand light chain immunoglobulins can be obtained from a murine hybridomaof interest and engineered to contain non-murine (e.g., human)immunoglobulin sequences using standard molecular biology techniques.For example, to create a chimeric antibody, the murine variable regionscan be linked to human constant regions using methods known in the art(see e.g., U.S. Pat. No. 4,816,567). To create a humanized antibody, themurine CDR regions can be inserted into a human framework using methodsknown in the art (see e.g., U.S. Pat. No. 5,225,539, and U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,762; and 6,180,370).

In embodiments, an IL-6a described herein (e.g., an anti-IL-6 antibodyor derivative or fragment thereof) can specifically bind human IL-6. Inembodiments, the IL-6a can specifically bind to site II of IL-6 (e.g.,site II of human IL-6).

In some embodiments, an IL-6a antibody is a human monoclonal antibody.Such antibodies can be generated using transgenic or transchromosomicmice comprising portions of a human immune system rather than the mousesystem. These transgenic and transchromosomic mice include mice referredto herein as the HuMAb Mouse® and KM Mouse®, respectively, and arecollectively referred to herein as “human Ig mice” (e.g., See U.S. Pat.Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,397; 5,661,016; 5,814,318, 5,814,318; 5,874,299; and 5,770,429;U.S. Pat. No. 5,545,807; PCT Publication Nos.: WO 92/03918, WO 93/12227,WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962; and PCTPublication No. WO 01/14424).

In another aspect, human anti-IL-6 antibodies can be raised using amouse that carries human immunoglobulin sequences on transgenes andtranschromosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice aredescribed in detail in PCT Publication No. WO 02/43478.

Other transgenic animal systems expressing human immunoglobulin genesare available in the art and can be used to raise an antibody IL-6a. Forexample, an alternative transgenic system referred to as the Xenomouse™(Abgenix, Inc.) can be used; such mice are described in, for example,U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584; and6,162,963. Moreover, transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raisean antibody IL-6a. For example, mice carrying both a human heavy chaintranschromosome and a human light chain transchromosome are described intomizuka et al. (2000, Proc Natl Acad Sci USA 97:722-727). Humanmonoclonal antibodies can also be prepared using SCID mice into whichhuman immune cells have been reconstituted such that a human antibodyresponse can be generated upon immunization. Such mice are described in,for example, U.S. Pat. Nos. 5,476,996 and 5,698,767.

Phage Display Libraries

In some cases, an antibody IL-6a antibody or derivative or fragmentthereof is produced in a method that involves synthesizing a library ofhuman antibodies using phage, screening the library with an IL-6, e.g.,a human IL-6, or a fragment thereof, isolating phage that bind IL-6, andobtaining the antibody from the phage.

Recombinant human antibody IL-6a can also be isolated by screening arecombinant combinatorial antibody library. In general, the library is ascFv phage display library, generated using human VL and VH cDNAsprepared from mRNA isolated from B cells. Methods for preparing andscreening such libraries are known in the art. Kits for generating phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagne SurfZAP™ phage display kit, catalog no. 240612). Other methodsand reagents that can be used in generating and screening antibodydisplay libraries are known in the art (see, e.g., U.S. Pat. No.5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO 92/20791,WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al.,Bio/Technology 9:1370-1372 (1991); Hay et al., Hum Antibod Hybridomas3:81-85 (1992); Huse et al., Science 246;1275-1281 (1989); McCafferty etal., Nature 348:552-554 (1990); Griffths et al., EMBO J 12:725-734(1993); Hawkins et al., J Mol Biol 226:889-896 (1992); Clackson et al.,Nature 352:624-628 (1991); Gram et al., Proc Natl Acad Sci USA89:3576-3580 (1992); Garrad et al., Bio/Technology 9:1373-1377 (1991);Hoogenboom et al., Nuc Acid Res 19:4133-4137 (1991); and Barbas et al.,Proc Natl Acad Sci USA 88:7978-7982 (1991), all incorporated herein byreference.

In an example for isolating and producing human IL-6 antibodies with thedesired characteristics, a human IL-6 antibody is first used to selecthuman heavy and light chain sequences having similar binding activitytoward IL-6, using epitope imprinting methods described in PCTPublication No. WO 93/06213, incorporated herein by reference. Theantibody libraries used in this method are generally scFv librariesprepared and screened as described in PCT Publication No. WO 92/01047;McCafferty et al., Nature 348:552-554 (1990); and Griffiths et al., EMBOJ 12:725-734 (1993), all incorporated herein by regerence.

Once initial human VL and VH domains are selected, “mix and match”experiments are performed, in which different pairs of the initiallyselected VL and VH segments are screened for IL-6 binding to selectpreferred VL/VH pair combinations. To select for desirable features ofan IL-6a, the VL and/or VH segments of a selected pair can be randomlymutated. This in vitro affinity maturation can be accomplished, forexample, by amplifying VH and VL domains using PCR primers complimentaryto a CDR of one or both of the selected VH and VL domains, which primerscontain a random mixcture of the four nucleotide bases at certainpositions such that the resultant PCR products encode VH and VL segmentsinto which random mutations have been introduced into the VH and/or VL.Such randomly mutated VH and VL segments can be re-screened for bindingto IL-6, e.g., to site II of IL-6.

Following screening and isolation of an antibody IL-6a from arecombinant immunoglobulin display library, nucleic acids encoding theselected antibody can be recovered from the display package (e.g., fromthe phage genome) and subcloned into other expression vectors usingrecombinant DNA techniques known in the art. Such antibodies can befurther manipulated to produce an antibody fragment such as thosedescribed herein.

Pharmacokinetics (PK)

Testing for PK can be performed using methods known in the art. Onebarrier to determinations requiring the use of an animal, for exampledetermination of PK, is that human IL-6 has less than 50% homology withthat of some animals commonly used for such testing. One method oftesting PK is therefore to use a transgenic mouse expressing human IL-6.In some embodiments, a non-human primate is used to determine PK.

In some embodiments, an anti-IL6 antibody is mutated to alter its PK,e.g., by altering the pH sensitivity of FcRn binding. A method ofobtaining such mutations is described in the Examples. Accordingly, insome embodiments, the IL-6a has altered systemic PK compared to aparental IL-6a or a reference molecule. In some cases, the PK is notaltered or is improved in the vitreous. In some embodiments, the IL-6ahas the same or increased PK (e.g., an increased half-life) in thevitreous and reduced systemic PK (e.g., as assayed in a circulatoryfluid such as blood, plasma, lymph, serum, liver, kidney, other tissue,or other body fluid) compared to a parental IL-6a or a referencemolecule.

Models for Testing an IL-6 Antagonist

IL-6 antagonists can be tested in models of disease for IL-6 associateddelivery, particularly for the efficacy of treatment and limiteddeleterious effects on advantagious IL-6 properties. For example,uveitis can be tested in an experimental autoimmune uveitis model inrats or mice (Caspi, Invest Ophthalmol Vis Sci 52:1873; Agarwal et al.,900;443-69, 2012) using interphotoreceptor retinoid-binding protein(TRBP) in complete Freund's adjuvant (CFA) immunization. Other modelsinclude those known in the art for dendritic cell-induced uveitis,adoptive transfer of cultured effector T cells, spontaneous EAU in IRBPTCR Tg mice, endotoxin-induced uveitis, autoimmune uveoretinitis (Harutaet al., Invest Ophthalmol Vis Sci 53:3264 (2011); Yoshimura et al.,Rheumatology 48:347-354 (2009)). Other model systems that can be used toexamine the effects of an IL-6a disease are, for example, a choroidalneovascularization (CNV) model (Izumi-Nagai et al., Am J Pathol 170:6(2007); Krzystolik et al., Arch Ophthalmol 120:338 (2002)) and diabeticmodels such as those described in Kern et al. (Animal Models Of DiabeticComplications Consortium (P01 DK57733), Update Report (September2001-January 2004)). Animal models useful for testing an IL-6a inrheumatoid arthritis are known in the art, e.g., see Asquith et al. (EurJ Immunol 39:2040-4 (2009)) and Kollias et al. (Ann Rheum Dis 70:1357-62(2011).

Combination Therapies

In some embodiments, an IL-6a is administered in combination with asecond therapeutic entity. For example, an IL-6a is administered in atreatment regime that includes a VEGF inhibitor such as ranidizumab. Insome embodiments, an IL-6a is administered in a treatment regime thatincludes a PDGF inhibitor such as an anti-PDGF antibody or anti-PDGFreceptor antibody (e.g., imatinib.

Delivery of IL-6 Antagonist

IL-6 antagonist can be delivered locally, either in direct contact withor near a cell or tissue being targeted for IL-6 inhibition.Non-limiting examples of such delivery methods include injection,infusion, or implantation of a substance containing an IL-6 antagonist

In some embodiments, the IL-6a composition is administered as anophthalmic formulation. The methods can comprise administration of theIL-6a composition and an ophthalmically acceptable carrier. In someembodiments, the ophthalmic formulation is a liquid, semi-solid, insert,film, microparticle, or nanoparticle.

In some embodiments, the IL-6a composition is formulated for topicaladministration, e.g., to the eye. The topical formulation can be aliquid formulation or semi-solid, for example, a topical formulation caninclude an aqueous solution, an aqueous suspension, an ointment or agel. An ophthalmic IL-6a formulation can be topically applied to thefront of the eye, under the upper eyelid, on the lower eyelid and in thecul-de-sac. Typically, the ophthalmic formulation is sterile. An IL-6aophthalmic formulation can contain one or more pharmaceutical excipientssuitable for the preparation of ophthalmic formulations. Examples ofsuch excipients are preserving agents, buffering agents, chelatingagents, antioxidant agents and salts for regulating the osmoticpressure. Ophthalmic formulations, including both ointments andsuspensions, typically have a viscosity that is suited for the selectedroute of administration. In some embodiments, the ophthalmic formulationhas a viscosity of from about 1,000 to about 30,000 centipoise.

In some embodiments, the formulation is a liquid formulation comprisinga polymer. Such a polymer can be used to improve the bioavailability,raise viscosity, or reduce drainage from the eye of a liquidformulation. Suitable polymers include, but are not limited to, thosedescribed in Wagh et al. (Asian J Pharm, 2;12-17, 2008). In non-limitingexamples, the polymer is sodium hyaluronase, chitosan, a cyclodextrin(e.g., hydroxypropyl-β-cyclodextrin), polygalactoronic acid, xyloglucan,xanthan gum, gellan gum, a thiomer, a poly(ortho ester) (e.g., Einmahl,Adv Drug Deliv Rev 53:45-73, 2001), or a tamaring seed polysaccharide(e.g., Ghelardi et al., Antimicrob Agents Chemother 48:3396-3401, 2004).

In some embodiments, a formulation comprising a IL-6a composition forophthalmic delivery can comprise one or more of surfactants, adjuvants,buffers, antioxidants, tonicity adjusters, preservatives (e.g., EDTA,BAK (benzalkonium chloride), sodium chlorite, sodium perborate,polyquaterium-1), thickeners or viscosity modifiers (e.g., carboxymethylcellulose, hydroxymethyl cellulose, polyvinyl alcohol, polyethyleneglycol, glycol 400, propylene glycol hydroxymethyl cellulose,hydroxpropyl-guar, hyaluronic acid, and hydroxypropyl cellulose) and thelike. Additives in the formulation may include, but are not limited to,sodium chloride, sodium bicarbonate, sorbic acid, methyl paraben, propylparaben, chlorhexidine, castor oil, and sodium perborate.

In some embodiments, purified or deionized water is used in thecomposition. The pH can be adjusted by adding any physiologically andophthalmically acceptable pH adjusting acids, bases or buffers to withinthe range of about 5.0 to 8.5, e.g., pH 7.0, pH 7.3, pH, 7.4, or pH 7.5.Ophthalmically acceptable examples of acids include acetic, boric,citric, lactic, phosphoric, hydrochloric, and the like, and examples ofbases include sodium hydroxide, sodium phosphate, sodium borate, sodiumcitrate, sodium acetate, sodium lactate, tromethamine,trishydroxymethylamino-methane, and the like. Examples of salts andbuffers that can be used in a formulation include citrate/dextrose,sodium bicarbonate, ammonium chloride and mixtures of the aforementionedacids and bases.

In some embodiments, the osmotic pressure of the ophthalmic compositionmay be from about 10 milliosmolar (mOsM) to about 400 mOsM, for example,200 to 400 mOsM, or 220 to 370 mOsM. Generally, the osmotic pressure canbe adjusted using physiologically and ophthalmically acceptable salts orexcipients. In some embodiments, sodium chloride is included in aformulation, for example, sodium chloride is present in a formulation ina concentration ranging from 0.01% to 1% by weight, or from 0.05% to0.45% by weight, based on the total weight of the composition.Equivalent amounts of one or more salts made up of cations such aspotassium, ammonium and the like and anions such as chloride, citrate,ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate,bisulfate, sodium bisulfate, ammonium sulfate, and the like can also beused in addition to or instead of sodium chloride to achieveosmolalities within the desired range. In some embodiments, a sugar suchas mannitol, dextrose, sorbitol, glucose and the like is also used toadjust osmolality.

In some embodiments, the methods involve forming or supplying a depot ofthe agent in contact with the external surface of the eye. A depotrefers to a source of agent that is not rapidly removed by tears orother eye clearance mechanisms. This allows for continued, sustainedhigh concentrations of agent be present in the fluid on the externalsurface of the eye by a single application. In some embodiments, thedepot can retoaio for up to eight hours or more. In some embodiments,the ophthalmic depot formulation includes, but is not limited to,aqueous polymeric suspensions, ointments, and solid inserts.

In some embodiments, a semi-solid composition is a liquid formulationthat increases in viscosity upon application to the eye, typically dueto the presence of a polymer in the liquid formulation for which anincrease is viscosity occurs with a change in temperature, pH, orelectrolyte concentration. The polymer can be, for example,celluloseacetophthalate, polyacrytic acid, getlan gum, hyaluronase,chitosan, salts of alginic acid (e.g., sodium alginate), or a blockcopolymer of ethylene oxide and propylene oxide (e.g., Pluronic®, BASF;poloxamer). In some embodiment, the polyacrylic acid is cross-linkedacrylic acid (e.g., Carbopol®). In some embodiments, the semi-solidcomposition comprises a mixture of carbopol and a block copolymer ofethylene oxide and propylene oxide; a mixture of methyl cellulose andhydroxyethyl cellulose; or a mixture of polyethylene glycol and a blockcopolymer of ethylene oxide and propylene oxide.

In some embodiments, the IL-6a containing ophthalmic formulation is anointment or gel. In some embodiment, the ophthalmic formulation is anoil-based delivery vehicle. For example, the formulation can comprises apetroleum or lanolin base to which the IL-6a composition is added, (forexample at 0.1 to 2%), and excipients. Common bases can include, but arenot limited to, mineral oil, petrolatum and combinations thereof. Insome embodiments, the ointment is applied as a ribbon onto the lowereyelid.

In some cases, the ophthalmic composition is an ophthalmic insert. Forexample, the ophthalmic insert is biologically inert, soft,bio-erodible, viscoelastic, stable to sterilization after exposure totherapeutic agents, reisitant to infections from air borne bacteria,bio-erodible, biocompatible, and/or viscoelastic. In some embodiments,the insert comprises an ophthalmically acceptable matrix, e.g., apolymer matrix. The matrix is typically a polymer and the IL-6 acomposition is dispersed within the matrix or bonded to the polymermatrix. In some embodiments, the agent is slowly released from thematrix through dissolution or hydrolysis of a covalent bond. In someembodiments, the polymer is bioerodible (soluble) and the dissolutionrate thereof can control the release rate of the agent dispersedtherein. In another form, the polymer matrix is a biodegradable polymerthat breaks down such as by hydrolysis to thereby release the agentbonded thereto or dispersed therein. In further embodiments, the matrixand agent can be surrounded with an additional polymeric coating tofurther control release. In some embodiments, the insert comprises abiodegradable polymer such as polycaprolactone (PCL), an ethylene/vinylacetate copolymer (EVA), polyalkyl cyanoacrylate, polyurethane, a nylon,or poly(dl-lactide-co-glycolide) (PLGA), or a copolymer of any of these.In some cases, the agent is dispersed into the matrix material ordispersed amongst the monomer composition used to make the matrixmaterial, prior to polymerization. In some embodiments, the amount ofagent is from about 0.1 to about 50%, or from about 2 to about 20%. Thebiodegradable or bioerodible polymer matrix can be used so that thespent insert does not have to be removed from the eye. As thebiodegradable or bioerodible polymer is degraded or dissolved, the agentis released.

In further embodiments, the ophthalmic insert comprises a polymer,including, but are not limited to, those described in Wagh, et al.“Polymers used in ocular dosage form and drug delivery systems”. AsianJ. Pharm., pages 12-17 (January 2008), which is incorporated herein byreference, in its entirety. In some embodintents, the insert comprises apolymer selected from polyvinylpyrrolidone (PVP), an acrylate ormethacrylate polymer or copolymer (e.g., Eudragit® family of polymersfrom Rohm or Degussa), hydroxymethyl cellulose, polyacrylic acid,poly(amidoamine) dendrimers, poly(dimethylsiloxane), polyethylene oxide,poly(lactide-co-glycolide), poly(2-hydroyethylmethacrylate), polyvinylalcohol), or poly(propylene fumarate). In some embodiments, the insertcomprises Gelfoam®. In some embodiments, the insert is a polyacrylicacid of 450 kDa-cysteine conjugate.

The insert can comprise a core that contains the IL-6a composition andan outer tube (e.g., as described in U.S. Patent Pub. No. 20040009222).In some cases, the outer tube can be permeable, semi-permeable, orimpermeable to the drug. In some embodiments, the core includes apolymer matrix that dees not have a significant effect on the rate ofIL-6a composition release. In some cases, the outer tube, the polymermatrix of the core, or both is bioerodible. The co-extruded product canbe segmented into drug delivery devices. In some embodiments, the deviceis uncoated so that the respective ends are open, or the device iscoated with, for example, a layer that is permeable to the IL-6acomposition, semi-permeable to the IL-6a composition, or bioerodible. Incertain embodiments, the IL-6a composition and at least one polymer areadmixed in powder form.

In some embodiments, the ophthalmic composition is an ophthalmic film.Polymers suitable for such films include, but are not limited to, thosedescribed in Wagh, et al. (supra). In some embodiments, the film is asoft-contract lens, for example, a lens composed of copolymers ofN,N-diethylacrylamide and methacrylic acid cross-linked withethyleneglycol dimethacrylate.

In certain embodiments, the IL-6a is in an insert that is in a tubularform, and may be segmented.

In some embodiments, the IL-6a composition is formulated in atherapeutically effective amount, coated by or dispersed in a polymermatrix, such that the IL-6a composition is in granular or particulateform. In some embodiments, the IL-6a composition is released from theformulation as drug from the granules dissolves into or within thematrix, diffuses through the matrix, and is released into thesurrounding physiological fluid. In some embodiments, the rate ofrelease is limited primarily by the rate of dissolution of the IL-6acomposition from the granules/particles into the matrix; the steps ofdiffusion through the matrix and dispersion into the surrounding fluidare primarily not release-rate-limiting. In certain embodiments, thepolymer matrix is non-bioerodible, while in other embodiments it isbioerodible. Exemplary non-bioerodible polymer matrices can be formedfrom polyurethane, polysilicone, poly(ethylene-co-vinyl acetate) (EVA),polyvinyl alcohol, and derivatives and copolymers thereof. Exemplarybioerodible polymer matrices can be formed from polyanhydride,polylactic acid, polyglycolic acid, polyorthoester,polyalkylcyanoacrylate, and derivatives and copolymers thereof.

In some cases, the IL-6a composition is formulated in a collagenousmaterial. For example, the insert can be a soluble ophthalmic druginsert (e.g., a polymeric oval film that can be introduced in the upperconjuctival sac for drug delivery; an elliptical insert such as OCUSERT®(pilocarpine ocular therapeutic system, developed by Alza Corporation)which is made of ethylene vinyl acetate; Lacrisert®, a rod shaped insertmade of cellulose; New Ophthalmic Drug Delivery Systems (NODS), made ofpoly(vinyl alcohol); or inserts such as those described in Fabrizio (AdvDrug Deliv Rev 16:95-106, 1998). In some cases, the insert comprisescollagen, gelatin, or a polymer, wherein the polymer is selected frompolycaprolactone (PCL), an ethylene/vinyl acetate copolymer (EVA),polyalkyl cyanoacrylate, polyurethane, a nylon,poly(dl-lactide-co-glycolide) (PLGA), or a copolymer of any of these. Insome cases, the insert is implanted under the upper eyelid. In somecases, the insert is implanted in the posterior segment of the eye, inthe choroidal space, or in the sclera. In some embodiments, the insertis implanted intravitreally or sub-retinally. In some embodiments, theinsert is injected sub-retinally. Methods of administration andtechniques for their preparation are set forth in Remington's: ThePractice of Science of Pharmacy, 20^(th) edition (Lippincott Williams &Wilkins, 2006) which is incorporated herein by reference in itsentirety.

In other embodiments, an insert containing an IL-6a composition providesa sustained release of the agent to the vitreous of the eye. As usedherein, “sustained release” means that the composition releases theagent over an extended period of time in a controlled fashion. In someembodiments, the insert releases the agent at a rate such that theaqueous agent concentration remains less than the vitreous agentconcentration during the release. In some embodiments, the aqueous agentconcentration is from about 0.002 μg/mL to about 0.01 μg/mL, or fromabout 0.01 μg/mL, to about 0.05 μg/mL, or less than about 0.05 μg/mL. Insome embodiments, the agent is released at a rate of about 1 μg/day toabout 50 μg/day, or from about 1 μg/day to about 10 μg/day. In someembodiments, the insert further comprises an additional therapeuticagent, as detailed above, e.g., fluocinolone acetonide (such as thatfound in the ophthalmic insert Retisert®).

In some embodiments, the ophthalmic composition comprises microspheresor nanoparticles. In some embodiment, the microspheres comprise gelatin.In some embodiments, the microspheres are injected to the posteriorsegment of the eye, in the choroidal space, in the sclera,intravitreally or sub-retinally. In some embodiments, the microspheresor nanoparticles comprises a polymer including, but not limited to,those described in Wagh, et al. (Asian J Pharm 2:12-17, 2008). In someembodiments, the polymer is chitosan, a polycarboxylic acid such aspolyacrylic acid, albumin particles, hyaluronic acid esters,polyitaconic acid, poly(butyl)cyanoacrylate, polycaprolactone,poly(isobutyl)caprolactone, poly(lactic acid-co-glycolic acid), orpoly(lactic acid). In some embodiments, the microspheres ornanoparticles comprise solid lipid particles.

In some embodiments, an IL-6a composition comprises an ion-exchangeresin. In some embodiments, the ion-exchange resin is an inorganiczeolite or synthetic organic resin. In some embodiments, theion-exchange resin includes, but is not limited to, those described inWagh, et al., supra, which is incorporated herein by reference in itsentirety. In some embodiments, the ion-exchange resin is a partiallyneutralized polyacrylic acid.

An IL-6a composition can be provided in an aqueous polymeric suspension.In some embodiments, the IL-6a composition or a polymeric suspendingagent is suspended in an aqueous medium (e.g., having the properties asdescribed above). Examples of polymeric suspending agents include, butare not limited to, dextrans, polyethylene glycols, polyvinylpyrolidone,polysaccharide gels, Gelrite®, cellulosic polymers like hydroxypropylmethylcellulose, and carboxy-containing polymers such as polymers orcopolymers of acrylic acid, as well as other polymeric demulcents. Insome embodiments, the polymeric suspending agent is a water swellable,water insoluble polymer, especially a cross-linked carboxy-containingpolymer. In some embodiments, the polymeric suspending agent comprisesfrom at least about 90% to about 99.9%, or from about 95% to about99.9%, by weight based on the total weight of monomers present, of oneor more carboxy-containing monoethylenically unsaturated monomers. Insome embodiments, the carboxy-containing monoethylenically unsaturatedmonomer includes acrylic acid, methacrylic acid, ethacrylic acid,methylacrylic acid (crotonic acid), cis-.alpha.-methylcrotonic acid(angelic acid), trans-α-methylcrotonic acid (tiglic acid),α-butylcrotonic acid, .alpha.-phenylacrylic acid, α-benzylacrylic acid,α-cyclohexylacrylic acid, phenylacrylic acid (cinnamic acid), coumaricacid (o-hydroxycinnamic acid), and umbellic acid (p-hydroxycoumaricacid). In some embodiments, the polymer is cross-linked by apolyfunctional crosslinking agent (e.g., a difunctional crosslinkingagent). In some embodiments, the crosslinking agent is contained its anamount of from about 0.01% to about 5%, or from about 0.1% to about5.0%, or from about 0.2% to about 1%, based on the total weight ofmonomers present. In some embodiments, the crosslinking agents arenonpolyalkenyl polyether difunctional crosslinking monomers such asdivinyl glycol, 2,3-dihydroxyhexa-1,5-diene, 2,5-dimethyl-1,5-hexadiene,divinylbenzene, N,N-diallylacrylamide, N,N-diallymethacrylamide;polyalkenyl polyether crosslinking agents containing two or more alkenylether groupings per molecule, e.g., alkenyl ether groupings containingterminal H₂C═C groups, prepared by etherifying a polyhydric alcoholcontaining at least four carbon atoms and at least three hydroxyl groupswith an alkenyl halide such as allyl bromide or the like, e.g.,polyallyl sucrose, polyallyl pentaerythritol, or the like; diolefinicnon-hydrophilic macromeric crosslinking agents having molecular weightsof from about 400 to about 8,000, such as insoluble diacrylates andpolyacrylates and methacrylates of diols and polyols, diisocyanatehydroxyalkyl acrylate or methacrylate reaction products of isocyanateterminated prepolymers derived from polyester diols, polyether diols orpolysiloxane diols with hydroxyalkylmethacrylates, and the like.

In some embodiments, the cross-linked polymers are made from acarboxy-containing monoethylenically unsaturated monomer or monomers asthe sole monoethylenically unsaturated monomer present, together with acrosslinking agent or agents. In some embodiments, the polymers are onesin which up to about 40%, and preferably from about 0% to about 20% byweight, of the carboxy-containing monoethylenically unsaturated monomeror monomers has been replaced by one or more non-carboxyl-containingmonoethylenically unsaturated monomer or monomers containing onlyphysiologically and ophthalmically innocuous substituents, includingacrylic and methacrylic acid esters such as methyl methacrylate, ethyl,acrylate, butyl acrylate, 2-ethylhexylacrylate, octyl, methacrylate,2-hydroxyethylmethacrylate, 3-hydroxypropylacrylate, and the like, vinylacetate. N-vinylpyrrolidone, and the like (e.g., Mueller et al. U.S.Pat. No. 4,548,990). In some embodiments, the polymers includepolycarbophil (Noveon AA-1), Carbopol®, and DuraSite®. In someembodiments, the cross-linked polymers are prepared by suspension oremulsion polymerizing the monomers, using conventional free radicalpolymerization catalysts, to a dry particle size of not more than about50 μm in equivalent spherical diameter. In some embodiments, the averagedry particle size is from about 1 to about 30 μm, or from about 3 toabout 20 μm in equivalent spherical diameter. In some embodiments, thepolymer particles are obtained by mechanically milling larger polymerparticles. In further embodiments, such polymers will have a molecularweight from about 250,000 to about 4,000,000, and from 3,000,000,000 to4,000,000,000. In other embodiments, the particles of cross-linkedpolymer are monodisperse, meaning that they have a particle sizedistribution such that at least about 80%, about 90% or about 95%, ofthe particles fall within a μm band of major particle size distribution.In further embodiments, the monodisperse particle size means that thereis no more than about 20%, about 10%, or about 5% particles of a sizebelow 1μm. In some embodiments, the aqueous polymeric suspensioncomprises from about 0.05 to about 1%, from about 0.1 to about 0.5%, orfrom about 0.1 to about 0.5%, of the agent and from about 0.1 to about10%, from about 0.5 to about 6.5%, from about 0.5 to about 2.0%, fromabout 0.5% to about 1.2%, from about 0.6 to about 0.9%, or from about0.6 to about 0.8% of a polymeric suspending agent. Although referred toin the singular, it should be understood that one or more species ofpolymeric suspending agent can be used with the total amount fallingwithin the stated ranges. In one embodiment, the amount of insolublelightly cross-linked polymer particles, the pH, and the osmotic pressurecan be correlated with each other and with the degree of crosslinking togive a composition having a viscosity in the range of from about 500 toabout 100,000 centipoise, and preferably from about 1,000 to about30,000 or about 1,000 to about 10,000 centipoise, as measured at roomtemperature (about 25° C.) using a Brookfield Digital LVT Viscometerequipped with a number 25 spindle and a 13R small sample adapter at 12rpm. In some embodiments, the viscosity is from about 10 to about 400centipoise, from about 10 to about 200 centipoises or from about 10 toabout 25 centipoise.

In some embodiments, the aqueous polymeric suspensions may be formulatedso that they retain the same or substantially the same viscosity in theeye that they had prior to administration to the eye. In someembodiments, they may be formulated so that there is increased gelationupon contact with tear fluid. For instance, when a formulationcontaining DuraSite® or other similar polyacrylic acid-type polymer isadministered to the eye at a pH of less than about 6.7 , the polymer mayswell upon contact with tear fluid since it has a higher pH (around 7).This gelation or increase in gelation may lead to entrapment of thesuspended particles, thereby extending the residence time of thecomposition in the eye. In some embodiments, the agent is releasedslowly as the suspended particles dissolve over time. In someembodiments, this delivery route increases patient comfort and increasedagent contact time with the eye tissues, thereby increasing the extentof drug absorption and duration of action of the formulation in the eye.The agents contained in these drug delivery systems will be releasedfrom the gels at rates that depend on such factors as the drug itselfand its physical form, the extent of drug loading and the pH of thesystem, as well as on any drug delivery adjuvants, such as ion exchangeresins compatible with the ocular surface, which may also be present.

In some embodiments, an IL-6 antagonist is provided to a subject usinggenetic delivery, e.g., local genetic delivery. such delivery can be viaa transient expression system, a stable (e.g., integrated) expressionsystem such as a lentiviral delivery system manufactured by Bluebird Bio(Cambridge, Mass.), or delivery in a cell factory such as thosemanufactured by Neurotech (Cumberland, R.I.).

All technical features can be individually combined in all possiblecombinations of such features.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein.

EXAMPLES

The following non-limiting examples further illustrate embodiments ofthe inventions described herein.

Example 1 Validation of Local IL-6 Blockade in ChoroidalNeovascularization (CNV) Model

To determine whether local IL-6 blockade could be effective for treatingeye disease, e.g., diabetic macular edema (DME) or wet AMD, an anti-IL-6antibody was locally administered using a model system for choroidalneovascularization. A laser-induced CNV model(eyecro.com/in-vivo/laser-induced-choroidal-neovascularization-cnv/)reproduces many of the pathologic processes underlying DME includinginflammation and angiogenesis. Studies were performed in rats at EyeCRO(Oklahoma City, OKla.). Six animals in each group underwent bilaterallaser treatment on Day 0 to produce three lesions per eye. On days 3 and10, 3 μg of a polyclonal anti-rat-IL-6 antibody (R&D Systems AF506;Minneapolis, Minn.) was administered to the test group by intravitreal(IVT) injection, while PBS or an anti-VEGF polyclonal antibody (R&DSystems AF564) was administered to the vehicle and positive controlgroups, respectively. In vivo angiography was performed on days 15 and22 to measure the lesion area. On both days 15 and 22, the anti-IL-6treated group had significantly reduced neovascularization compared tothe vehicle control. There was no significant difference in responsebetween the anti-IL-6 treated group and the anti-VEGF positive control.FIG. 1 shows the results of such an experiment. These data demonstratethat an IL-6a, e.g., an anti-IL6 antibody, administered IVT can reduceneovascularization in a rat CNV model to similar levels as an anti-VEGFpositive control (p=0.0054 on Day 15 and p=0.0005 on Day 22 foranti-IL-6 vs. vehicle control).

These data indicate that local blockade of IL-6 can be useful fortreating eye disease such as diseases involving vascular leakage, e.g.,macular edema.

Example 2 Candidate Antibody IL-6 Antagonists

Candidate antibody IL-6 antagonists were developed using a process thatfirst involved immunizations. Immunizations were performed at thedirection of the inventors by a contract research organization (CRO).Five BALB/C mice were injected subcutaneously with 80 μg human IL-6 (R&DSystems, cat# 206-IL/CF, Minneapolis, Minn.) in PBS containing 1 M NaClwith Freud's adjuvant. Two boosts were performed with 80 μg and 50 μgIL-6. Spleen cells were harvested from the highest titer mouse and fusedwith P3x763Ag8.653 myeloma cells to form hybridomas.

Hybridoma supernatants were screened for IL-6 binding and antagonism.For the binding ELISA, Costar 9018 plates were coated with 1 μg/mL humanIL-6 in PBS overnight at 4° C. Wells were blocked with PBS containing 2%BSA, washed, and then incubated with 50 μL of each hybridoma supernatantdiluted 1:2 with PBS containing 2% BSA. After 60 minutes, wells werewashed three times with 300 μl PBS containing 0.1% Tween-20.Anti-mouse-HRP diluted 1:3000 in PBS-BSA was then added to each well andincubated for 30 minutes. Wells were washed as above then3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added and the signalmeasured at 450 and 550 nm. For antagonism studies, HEK-Blue™-IL6reporter cells (InvivoGen. San Diego, Calif.) were incubated withincreasing concentrations of human IL-6 in the presence of 1:10 dilutedhybridoma supernatant. After 20-24 hours, 20 μl of supernatant was mixedwith 180 μl QuantiBlue™ (InvivoGen) and the absorbance measured at 655nm.

Based on binding and antagonism studies, hybridoma 64 was selected byapplicants as a lead and subcloned at the CRO. Hybridoma 64 (a murinemonoclonal) was further tested for the ability to inhibit binding ofIL-6/IL-6Rα complex to gp130 using an enzyme-linked immunosorbant assay(ELISA). Hybridoma 64 at a concentration of 1.5 μg/ml significantlyreduced binding of an IL-6/IL-6Rα complex to immobilized gp130 by ELISA(FIG. 2).

The subclones were rescreened and the variable domains of subclone 64.58were amplified by 5′ RACE PCR and sequenced. The mouse variable domainsequences (referred to as m64) are as follows:

m64 VH (variable heavy chain) (SEQ ID NO: 13)QVQLQQSGAELVRPGTSVKVSCKASGYAFSNYLIEWVKQRPGQGLEWIGVITPGSGTINYNEKFKGKAVLTADKSSSTVYMQLSSLTSDDSAVYFCAKSRWDPLYYYALEYWGQGTSVTVSS m64 VL (variable light chain) (SEQ ID NO: 14)DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPL TFGAGTKLELK

To create humanized sequences, the m64 complementarity determiningregions (CDRs) were grafted into a human germline framework selected forsimilarity to the mouse sequence by a computational algorithm. Thehumanized sequences (referred to as h64) were as follows (alteredresidues compared to the m64 sequences are underlined) and have about79.5% identity (VH) and 84.4% identity (VL) with the murine sequences:

h64 VH (SEQ ID NO: 15)QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGVITPGSGTINYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSRWDPLYYYALEYWGQGTTVTVSS h64 VL (SEQ ID NO: 16)DIVMTQSPDSLAVSLGERATINCRASESVDNYGISFMNWYQQKPGQPPKLLIYAASNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPL TFGQGTKLEIKThe humanized sequences were synthesized by DNA2.0 (Menlo Part, Calif.),then cloned into pcDNA3.1 derived expression vectors as inline fusionswith the human IgGI constant domains. IgGs were expressed by transienttransfection in Freestyle™-293 cells (Invitrogen, Grand Island, N.Y.)and purified by protein-A chromatography. In both binding and antagonismstudies, the h64 IgG demonstrated considerably reduced potency comparedto its m64 predecessor. Therefore, yeast display was utilized to restorethe lost affinity.

To carry out the affinity maturation designed to restore or improve theaffinity of the humanized h64IgG, the h64 antibody sequences wererecloned to generate a Fab molecule in pYC2/CT-derived yeast vectors inwhich the FabH chain was fused to the anti-FITC scFv 4m5.3 through a(G4S)3 linker. A library of h64 variants was then generated by errorprone PCR following the protocol of Chao et al. (2006, Nature Protocols,1:755-768). H64 variants were expressed and surface captured by yeastlabeled with FITC-PEG-NHS then incubated with biotinylated human IL-6.Bound IL-6 was detected with streptavidin-APC, and cells with thehighest amount of bound IL-6 relative to the amount of displayed Fabswere selected on a BD FACSAria™ cell sorter. After four rounds ofselection, a population of higher affinity variants was selected andsequenced. The sequence of the clone selected by affinity maturation(referred to as h64-1.4) is as follows with the selected mutations(i.e., mutated compared to the sequences of h64 VH and VL) in boldfaceand the CDRs are underlined. These are the variable domains of 018 (aswell as the 020 and 029 IL-6a molecules described below). Note that thefull Fabs include the CK and IgGl CHl domains. In the context of thisapplication, reference to a “Fab” heavy chain or light chain amino acidsequence means that sequence can be part of a functioning Fab consistingof a light chain-derived sequence and a heavy chain-derived sequence.

h64-1.4 VH (018VH)(variable domain) (SEQ ID NO: 17)QVQLVQSGAEVKKPGSSVKVSCKASGYALSNYLIEWVRQAPGQGLEWMGVITPGSGTINYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSRWDPLYYYALEYWGQGTTVTVSS h64-1.4 VL(018VL)(varible domain) (SEQ ID NO: 18)DIVMTQSPDSLAVSLGERATINCRASESVDNYGIPFMNWYQQKPGQPPKLLIYAASNRGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSEEVPL TFGQGTKLEIKRTVThe h64-1.4 variable domains were recloned into the pcDNA3.1 human IgG1vector and expressed as a full length IgG1 in Freestyle™-HEK293 cells(Life Technologies). The resulting purified IgG was significantly morepotent than the original h64 antibody in both binding and cellularantagonism studies. Testing affinity using the yeast system, theaffinity increased from 343 pM for the original humanized molecule to 43pM. The antagonist potency was about a ten-fold increase as assayedusing the HEK-Blue cell system.

The h64-1.4 IgG was reformatted as a Fab for use in ocular and otherindications. Additionally, another round of library generation and yeastbased selections was performed to further improve affinity. After fourrounds of selection, there was significant enrichment for a VH variantwith the A79V mutation. Antibodies, variants and fragments thereofcomprising the A79V variant are referred to as 019 IL-6a antibodies,variants, and fragments thereof.

Example 3 Format Selection

To investigate suitable formats for an antibody-based IL-6 antagonist,IL-6 antibodies selected as described supra were tested for transientexpression, stability, aggregation properties, binding affinity, andIC50 using Fab, scFv (V_(B)-V_(L)) and scFv(V_(L),V_(B)) forms of the018 sequences.

Results of these studies for one of the candidate IL-6a molecules(sequences containing the 018 variable region are shown in Table 1.

TABLE 1 Parameter Fab scFv(V_(B)-V_(L)) scFv(V_(L-)V_(H)) Transientexpression 45 mg/ml 2 mg/L 4 mg/L Stability (T_(M)) 73° C. 43° C. 46° C.Aggregation (SEC, No Yes N/A MALS) Binding affinity (K_(D)) 240 pM 1 nM720 pM IC50 with 10 pM IL-6 255 pM 160 pM 125 pMThese data demonstrate a method of identifying key features of variousformats of an antibody-based IL-6 antagonist and illustrates that forIL-6 antagonists containing the 018 variable regions, the 018Fab formathas the most favorable features in most key categories, i.e.,expression, stability, aggregation, and binding affinity compared to anscFv configuration. The IC50 of the 018 Fab falls within a reasonablerange for therapeutic use.

Example 4 Examples of IL-6a Antibodies, Fragments, and Derivatives

Applicants have identified the following sequences using methodsdescribed herein. Underlined sequences represent CDRs of the heavy andlight chains. Other sequences can be found throughout the specification.

018 Heavy chain (full length; fl018HC) polypeptide sequence in an IgG1 framework(SEQ ID NO: 19)QVQLVQSGAEVKKPGSSVKVSCKASGYALSNYLIEWVRQAPGQGLEWMGVITPGSGTINYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSRWDPLYYYALEYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK018 Heavy chain (full length; fl018HC) nucleic acid sequence in an IgG1 framework(SEQ ID NO: 20)CAAGTGCAGCTGGTGCAGTCAGGGGCCGAGGTTAAGAAGCCAGGGAGCAGCGTCAAGGTATCTGTAAAGCGTCTGGTTACGCCCTTTCAAACTACCTGATCGAATGGGTGAGGCAGGCTCCCGGCCAAGGCCTGGAATGGATGGGAGTTATCACCCCTGGGAGCGGCACCATTAATTACGCCCAGAAATTTCAGGGACGAGTGACGATTACCGCCGACCAGTCCACCAGTACTGCCTACATGGAGCTGTCCTCACTCCGCAGCGAGGACACGGCAGTTTACTACTGCGCCCGGAGTCGTGGGACCCTCTTTACTATTATGCTCTGGAATACTGGGGCCAGGGAACGACCGTTACAGTGTCATCTGCTAGCACAAAAGGACCATCAGTCTTCCCACTTGCTCCTTCATCTAAGAGCACAAGTGGTGGCACTGCAGCCCTTGGCTGCCTGGTGAAAGATTATTTCCCCGAACCTGTTACAGTTTCTTGGAACTCCGGTGCACTGACATCCGGAGTACACACTTTCCCAGCTGTGCTGCAGAGCTCAGGACTGTATAGCCTGTCTTCGGTGGTCACTGTTCCATCGTCGAGTCTTGGCCACAGACATATATTTGCAACGTCAATCACAAGCCCTCCAACACAAAAGTGGATAAGAAGGTCGAGCCCAAATCTTGTGACAAGACCCATACGTGTCCTCCCTGTCCCGCCCCTGAACTGCTGGGAGGCCCTTCTGTGTTCCTGTTCCCACCTAAGCCAAAGGACACTCTGATGATCAGCCGGACTCCCGAGGTTACCTGTGTGGTGGTGGATGTGTCTCATGAAGACCCTGAGGTTAAGTTCAATTGGTACGTGGATGGCGTCCGAGGTGCATAACGCAAAAACCAAGCCGAGAGAGGAGCAGTACaatAGCACCTATAGAGTAGTGAGCGTCCTGACTGTCTTACATCAGGATTGGCTCAATGGTAAAGAATATAAGTGCAAGGTAAGCAACAAGGCCCTACCCGCACCAATAGAGAAGACCATCTCCAAGGCGAAAGGTCAGCCCAGGGAGCCCCAGGTTTATACATGCCTCCCTCACGCGACGAATTAACAAAGAATCAGGTGTCTCTCACCTGTCTCGTCAAGGGCTTTTACCCTTCCGACATCGCGTGGAGTGGGAATCCAATGGCCAGCCTGAGAACAATTATAAGACAACTCCCCCCAGTCCTGGATTCAGATGGGTCGTTCTTTCTATATAGTAAGTTGACCGTGGATAAGTCTCGCTGGCAACAGGGGAACGTGTTCTCTTGCTCTGTTATGCATGAAGCGCTGCACAATCATTATACCCAGAAGTCCCTGTCCCTGAGCCCCGGGAAG018 Fab Heavy Chain (018FabHC) polypeptide sequence in an IgG1 framework.CDRs are underlined (SEQ ID NO: 1)QVQLVQSGAEVKKPGSSVKVSCKASGYALSNYLIEWVRQAPGQGLEWMGVITPGSGTINYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCRSRWDPLYYYALEYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC018 full lenght chain (fl018LC)polypeptide sequence. CDRs are underlined(SEQ ID NO: 2)DIVMTQSPDS LAVSLGERAT INCRASESVD NYGIPFMNWY QQKPGQPPKL LIYAASNRGSGVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQQSEEVPL TFGQGTKLEI KRTVAAPSVFIFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLSSTLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC018 full length light chain (018LC)nucleic acid sequence in an IgG1 framework(SEQ ID NO: 26) GACATAGTGA TGACTCAAAG TCCGGACAGC CTGGCGGTGT CACTCGGCGAACGGGCAACT ATCAACTGCC GAGCCAGCGA GAGCGTCGAT AATTACGGCA TCCCCTTCATGAACTGGTAT CAGCAGAAGC CAGGACAGCC GCCCAAGCTG CTTATCTACG CCGCTTCCAACCGGGGATCA GGGGTGCCCG ATCGATTTAG TGGAAGCGGT AGTGGGACCG ATTTCACACTGACCATCAGC TCCCTTCAGG CCGAGGATGT GGCTGTCTAT TATTGTCAGC AATCCGAGGAAGTGCCGCTC ACGTTTGGTC AGGGAACCAA ACTGGAGATC AAGCGGACCGTAGCGGCGCC TAGTGTCTTC ATCTTCCCAC CCTCCGACGA ACAGCTGAAG TCTGGCACTGCTTCCGTCGT GTGCCTGCTC AACAACTTTT ACCCTAGAGA GGCAAAAGTT CAATGGAAAGTAGACAATGC CTTGCAGTCC GGGAACTCCC AGGAGTCTGT CACAGAGCAGGATAGTAAGG ACTCAACCTA CAGCCTGTCC AGCACACTGA CCCTCTCCAA AGCCGACTACGAGAAGCACA AAGTGTACGC TTGCGAAGTT ACGCATCAGG GGCTGTCCTC ACCCGTTACAAAAAGTTTTA ACAGAGGGGA GTGC019 Fab Heavy Chain (019FabHC, sequence as 018FabHC except for A79V (bold/italic)(SEQ ID NO: 3)QVQLVQSGAE VKKPGSSVKV SCKASGYALS NYLIEWVRQA PGQGLEWMGV ITPGSGTINYAQKFQGRVTI TADESTSTVY MELSSLRSED TAVYYCARSR WDPLYYYALE YWGQGTTVTVSSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQSSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSC019 VH (variable region/019HC, same sequence as 018HC variable region exceptfor A79V (bold/italic) (SEQ ID NO: 27)QVQLVQSGAE VKKPGSSVKV SCKASGYALS NYLIEWVRQA PGQGLEWMGV ITPGSGTINYAQKRQGRVTI TADESTSTVY MELSSLRSED TAVYYCARSR WDPLYYYALE YWGQGTTVT SS

The 019 antibody light chain (019LC) sequence (polypeptide and nucleicacid) is the same as the 018LC

CDR1 of 018HC (VH CDR1 018): (SEQ ID NO: 4) GYALSNYLIE CDR2 of 018HC (VH CDR2 018): (SEQ ID NO: 5) VITPGSGTIN CDR3 of 018HC (VH CDR3 018): (SEQ ID NO: 6) SRWDPLYYYALEY CDR1 of 018LC (VL CDR1): (SEQ ID NO: 7) RASESVDNYGIPFMN CDR2 of 018LC (VL CDR2): (SEQ ID NO: 8) AASNRGS CDR3 of 018LC (VL CDR3): (SEQ ID NO: 9) QQSEEVPLT CDR1 of 019HC (VH CDR1 019): (SEQ ID NO: 4) GYALSNYLIE CDR2 of 019HC (VH CDR2 019): (SEQ ID NO: 5) VITPGSGTIN CDR3 of 019HC (VH CDR3 019): (SEQ ID NO: 6) SRWDPLYYYALEY 

Example 5 Epitope and Structure Mapping

Epitope Mapping

Functional epitope mapping was performed on selected candidate IL-6antagonists. It was found that a candidate antibody (murine 64 antibody)did not reduce binding of IL-6Rα to IL-6 in an ELISA indicating that thecandidate antibody is not binding to site I. Additional experiments wereconducted demonstrating that chimeric murine 64 antibody reduced bindingof IL-6/IL-6Rα complex to gp130 in an ELISA indicating that either SiteII or Site III of IL-6 harbored the binding site for the antibody. Itwas also found that murine 64 antibody did not significantly blockbinding of a known site III binding antibody AH-65 (Immunotech,Marseille, France) to IL-6 indicating that the candidate antibody bindssite II of IL-6. These data demonstrate that antibodies against site IIcan be generated and demonstrates a method of identifying suchantibodies.

To further define the epitope, mutations in IL-6 were generated in yeastas fusions to 4m5.3 (Boder et al., 2000, Proc Natl Acad Sci USA 97,10701-10705; Chao et al., 2006, Nat Protoc 1, 755-768). The mutationsexpressed were in human IL-6 with the following single or doublemutations: R24E/D27E, R30E, Y31E, D34R, S118R/V121E, W157E, Q159E/T162P,K171E, and R179E. The expressed mutated IL-6 molecules were used inbinding studies with 018 (Fab). Reduced affinity for 018 (Fab) wasobserved for R24E/K27E, Y31E, D34R, and S118R/V121R, all of which arelocated in site II of IL-6. Accordingly, the invention described hereinincludes an antibody that binds to at least one, two, three, four, five,or six of the amino acids at position 24, 27, 31, 34, 118, and 121 ofhuman IL-6 or the equivalent site in an IL-6.

Structural Definition of a Site II Epitope

The following distances were calculated to structurally define site II.The calculations are based on the IL-6/IL-6α/gp130 hexameric crystalstructure, PDB 1P9M (Boulanger et al., 2003, Science 300:2101-2104).Helix 1 of IL-6 runs between site I and site II resulting in certainresidues that fall close to site II but have side chains that pointtoward site I, e.g., R30, D2 and D3 refer to extracellular domains ofIL-6Rα.

The following amino acids of IL-6 were determined to fall within 5 Å ofgp130-D2-D3: L19, R24, K27, Q28, R30, Y31, D34, E110, Q111, R113, A114,M117, S118, V121, Q124, F125, and K128

The following amino acids were determined to fall within 7 Å ofgp130-D2-D3: L19, E23, R24, I25, K27, Q28, I29, R30, Y31, D34, K41,Q102, E109, E110, Q111, A112, R113, A114, V115, Q116, M117, S118, K120,V121, L122, Q124, F125, and K128.

Accordingly, a molecule, e.g., an antibody or fragment thereof that canbind one or more of the IL-6 amino acids falling within 5 Å or 7 Å ofsite II can be an IL-6a.

The sequence of human IL-6 is provided below for reference (underlinedsequence is the leader sequence). Amino acids within 7 Å of gp130-D2-D3are in italics. The amino acid numbering, e.g., mutations used to defineepitopes, is without the leader sequence:

(SEQ ID NO: 21) MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKE FLQSSLRALRQM

Experiments were conducted testing the Fab fragment of the h64-1,4humanized antibody and demonstrated that it was able to block both cisand trans IL-6 signaling, which is due to site II targeting. The potencyof the Fab fragment was unchanged in the presence of soluble IL-6receptor (sIL-6R). This is in contrast to an anti-IL-6R IgG that haddecreased potency in the presence of sIL6R, and that blocks cissignaling only.

These experiments demonstrate that an antibody or fragment of theantibody such as a Fab fragment that targets site II can be used toinhibit both cis and trans signaling of IL-6.

Example 6 Primate Studies

Because non-primate activities can differ greatly from those ofprimates, candidate IL-6 antagonists are typically further assessed forPK and other parameters using non-human primates. Human IL-6 differsfrom cynomolgus monkey and rhesus monkey IL-6 at seven sites, one ofwhich is in site II (amino acid 28) and is the same at site II inAfrican green monkey IL-6. This appears to decrease binding of anantibody comprising 018 sequences by only about 3-4 fold. The ability tobind to a non-human primate IL-6 is a useful feature of an IL-6antagonist, facilitating development of the candidate as a drug, e.g.,by enabling testing such as toxicology testing in non-human primates.

As with most IL-6 antibodies, anti-IL-6 antibodies described herein didnot cross-react to rodent, rabbit, or canine IL-6 due to low sequencehomology. However, in affinity studies, it was found that 018 Fab bindscynomolgus monkey and African green monkey IL-6 with approximately humanaffinity (Table 2).

TABLE 2 Monovalent affinity (018 Fab) for various IL-6 of variousspecies Species K_(D) Human 200 pM African Green Monkey 280 pMCynomolgus monkey 840 Pm Dog >1 μM Mouse >1 μM Rabbit >1 μM Rat >1 μMThese data further demonstrate the ability of an IL-6 a as describedherein to specifically bind and the ability to develop a molecule havingfeatures permitting testing, e.g., for toxicology and reproductivestudies, in a suitable animal.

Example 7 Increasing Expression of an IL-6a

To increase expression of 018 Fab and 019 Fab polypeptides, constructswere made introducing five additional amino acids (DKTHT) to the heavychain in the CH1/hinge region using methods known in the art. Thesequence of the altered 018Fab heavy chain is shown below as SEQ IDNO:24. The altered 018 sequence is referred to herein as 020 and thealtered 019 sequence is referred to herein as 021. The 020 molecule (the020Fab heavy chain and the 018Fab light chain) had improved expressioncompared to the parent Fab that had 018Fab heavy and 018Fab lightchains. The 019 molecule exhibited no significant affinity differencecompared to the 020 molecule. Expression of both 020 and 019 wasincreased by about two fold, respectively, and the affinities were notaffected by the alteration.

020 Heavy chain (Fab with DKTHT at the carboxy terminus)(SEQ ID NOT: 24)QVQLVQSGAE VKKPGSSVKV SCKASGYALS NYLIEWVRQA PGPQLEWMGV ITPGSGTINYAQKFQGRVTI TAKESTSTAY MELSSLRSED TAVYYCARSR WDPLYYYALE YWGQGTTVTVSSASTKGPSV FPLAPSSKST SGGTAALGCK VKDYFPEPVT VSWNSGALTS GVHTFPAVLQSSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCDKTHT

IL-6 antagonism using the 020Fab was measured in HEK-Blue™ IL-6 reportercells (InvivoGen, San Diego, Calif.). Cells were incubated in a mixtureof 10 pM IL-6 and varying concentrations of either 020 or IL-6Rαantibody (Cell Sciences, Canton, Mass.), with or without 50 nM IL-6Rα.After 20-24 hours of incubation, 20 μL of cell culture supernatant wasmixed with 180 μL of QuantiBlue™ (InvivoGen) substrate and incubated forone hour; the absorbance was then measured at 655 nm. FIG. 3A and FIG.3B show data from these experiments, demonstrating the ability of 020 toinhibit IL-6 activity in the presence or absence of IL-6R.

Example 8 IgG2 IL-6 Antibodies

018 was reformatted into a human IgG2 isotype framework to reduce FcγRbinding and reduce ADCC compared to the IgG1 formatted antibody usingmethods known in the art. In addition, reformatting 018 to a full-lengthformat, e.g., an IgG2, is expected to decrease the rate of clearancefrom the vitreous due to the larger size of the molecule.

Construction/Purification of Anti-IL6 IgG2 Antibodies

To construct human IgG2 antibodies using anti-IL-6 sequences describedsupra, a human IgG2 constant domain was PCR amplified from cDNA withNheI and MluI restriction sites at the N- and C-terminal ends,respectively. The PCR product was purified, digested with NheI and MluIrestriction enzymes, and then ligated into pTT5 vector containinganti-IL6 variable domain, i.e., SEQ ID NO:1 (see above). This yielded afull-length IgG2 heavy chain sequence. Plasmids containing thefull-length light chain containing the 018 sequence were used to providelight chain.

To further reduce FcRn binding and thereby reduce recycling of theIL-6a, point mutations were made in the heavy chain. The mutations weremade by QuikChange® mutagenesis (Agilent Technologies, Santa Clara,Calif.). The heavy and light chain plasmids were co-transfected usingpoly(ethylenimine) (PEI) into 100 mL transient cultures of HEK293-6Ecells and cultured to allow expression for about five days. Thisgenerated antibodies containing an anti-IL-6 site II binding moiety andIgG2 structure. Such structures containing 018 CDRs are termed herein018IgG2 or 029. The point mutations were made at residues I253.

The IgG2 molecule was well expressed and blocks IL-6 in cellular assayswith slightly improved potency compared to the 020Fab.

029 mature sequences (CRSs underlined) 029 Heavy chain (SEQ ID NO: 11)QVQLVWSGAE VKKPGSSVKV SCKASGYALS NYLIEWVRQA PGQGLEWMGV ITPGSGTINYAQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCARSR WDPLYYYALE YWGQGTTVTVSSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQSSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV ERKCCVECPP CPAPPVAGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYV DGVEVHNAKT KPREEQFNSTFRVVSVLTVV HQDWLNGKEY KCKVSNKGLP APIEKTISKT KGQPREPQVY TLPPSREEMTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD SDGSFFLYSK LTVDKSRWQQGNVFSCSVMH EALHNHTQK SLSLSPGK 029 Light chain (SEQ ID NO: 12)DIVMTQSPDS LAVSLGERAT INCRASESVD NYGIPFMNWY QQKPGQPPKL LIYAASNRGSGVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQQSEEVPL TFGQGTKLEI KRTVAAPSVFIFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLSSTLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

Altered FcRn Binding

IL-6 can have certain positive systemic effects. It is therefore anadvantage to engineer an IL-6a that has good retention in the vitreousbut has a limited systemic half-life. The reduction or elimination ofFcRn binding should reduce systemic accumulation of any drug thatescapes into circulation, thereby improving safety of an IL-6a.

Accordingly, because FcRn mediated trafficking may increase the effluxof antibodies from the eye, the 020 IgG2 was further modified to ablateFcRn binding by introducing Fc mutations at residues I254, H311, or H436(See SEQ ID NO:23) numbering according to Martin et a., Molecular Cell7:4, 867-877 (2001)). The mutated sites are shown in boldface in SEQ IDNO:23; I254 was mutated to A or R, H311 was mutated to A or E, H311 wasmutated to N with D 313 mutated to T, and H436 was mutated to A(numbering starts after the leader sequence, which is underlined in SEQID NO:23. IL-6 antagonists containing such sequences are termed018IgG2m.

Antil-IL-6 heavy chain (IgG2) (regular font: VH; italic font: (CH) (withoutleader sequence) showing mutation sites (boldface) (SEQ ID NO: 23)QVQLVQSGAE VKKPGSSVKV SCKASGYALS NYLIEWVRQA PGQGLEWMGV ITPGSGTINYAQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCARSR WDPLYYYALE YWGQGTTVTVSSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQSSASTKGPSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV ERKCCVECPP CPAPPVAGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYV DGVEVHNAKT KPREEQFNSTFRVVSVLTVV HQDWLNGKEY KCKVSNKGLP APIEKTISKT KGQPREPQVY TLPPSREEMTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD SDGSFFLYSK LTVDKSRWQQGNVFSCSVMH HALHNHYTQK SLSLSPGKAnti-Il-6 heavy chain (IgG2) (regular font: VH; italic font: CH) with leadersequence (underlined) showing mutation sites (boldface) (SEQ ID NO: 24)MDWTWRILFLVAAATGAHSQVQLVQSGAE VKKPGSSVKV SCKASGYALS NYLIEWVRQAPGQGLEWMGV ITPGSGTINY AQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCARSRWDPLYYYALE YWGQGTTVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVTVSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTVERKCCVECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYVDGVEVHNAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEY KCKVSNKGLP APIEKTISKTKGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLDSDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK

Accordingly, some embodiments include an antibody having the heavy chainsequence depicted in SEQ ID NO:23 with mutations at I254 (e.g., A or R),h311 (mutated to A or E), H436 (mutated to A), or D313 (mutated to T)with H311 mutated to N.

SEQ ID NO:25 therefore provides a sequence that, when mutated at I133(e.g., I133A or I133R), H190 (e.g., H190A or H190E), H315 (e.g., H315A),or D192 with H190 (e.g., D192T with H190N) can be used in an antibody,fragment, or derivative thereof to produce a polypeptide having reducedFc binding at low pH, e.g., pH 5.5 or lysosomal pH and/or a polypeptidehaving reduced systemic half-life compared to a parent or otherreference molecule that does not include the sequence.

(SEQ ID NO: 25)SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQSSASTKGPSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV ERKCCVECPP CPAPPVAGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYV DGVEVHNAKT KPREEQFNSTFRVVSVLTVV HQDWLNGKEY KCKVSNKGLP APIEKTISKT KGQPREPQVY TLPPSREEMTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD SDGSFFLYSK LTVDKSRWQQGNVFSCSVMH HALHNHYTQK SLSLSPGKAnti-IL-6 light chain (IgG2) (regular font: VK; italic font: CK(SEQ ID NO: 22)DIVMTQSPDSLAVSLGERATINCRASESVDNYGIPFMNWYQQKPGQPPKLLIYAASNRGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSEEVPLTFGQGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADYEKHKVYACEV THQGLSSPVT KSFNRGEC

Example 9 Formulation Stability

The stability of the anti-IL-6/IgG1 Fab fragment (containing the IgG1CH1domain) was tested by determining the T_(m) initially in PBS then in arange of buffers and excipients using differential scanning fluorimetry.It was found that citrate buffer, pH 5.5 increased the T_(m) to morethan 80° C. Accordingly, in some embodiments, an IL-6a is provided incitrate buffer and in some cases has a T_(m) of at least 80°C.

Aggregation was tested using SEC-MALS and no aggregation was observed at20 mg/ml in phosphate buffered saline (PBS).

Example 10 pH Sensitive Antibodies for Enhanced PK

IL-6 can have certain positive systemic effects. It is therefore anadvantage to engineer an IL-6a that has good retention in the vitreousbut has a limited systemic half-life. The reduction or elimination ofFcRn binding should reduce systemic accumulation of any drug thatescapes into circulation, thereby improving safety of an IL-6a.Accordingly, because FcRn mediated trafficking may increase the effluxof antibodies from the eye, the 020 IgG2 was further modified to ablateFcRN binding by introducing Fc mutations at residues I253, h310, or H435(numbering according to Martin et al. (Molecular Cell, 7:4,867-877(2001)). Such antibodies are referred to herein as IL-6pH antibodies oranti-IL-6pH and are further described below.

Generation of IL-6ipH Antibodies

The pKa of histidine is about 6.0 and histidines inserted at bindinginterfaces can disrupt binding upon side-chain protonation at low pH.Using an anti-IL-6 site II targeted antibody as deseribed herein, alibrary was generated containing histidine-rich variants of CDRs from018 and the library was screened for pH-sensitive binding using yeastdisplay. The library generated was a combinatorial library with CDRsencoded by degenerate codons such that each residue is either awild-type residue (i.e., the same as in the parental antibody) or ahistidine residue. The screening was performed by alternating sortingfor high binding at physiological pH (7.4) and low binding at endosomalpH (5.5).

A yeast-selected mutant was identified that had relatively high bindingat pH 7.4 (monovalent Kd of 407 pM for the mutant compared to 192 pM forthe parent molecule) and relatively low binding at pH 5.5 (monovalent Kdof 2.362 nM for the mutant compared to 195 pM for the parent). Thisconstitutes an approximately 5.8 fold change in the affinity at pH 5.5.This mutant contained multiple histidine mutations in the light chainCDR1. Thus, the mutant demonstrated similar binding to the parentmolecule at pH 7.4, and a significant loss of affinity at pH 5.5. Thisobservation was verified using ELISA, FACS, and SPR analysis by methodsknown in the art.

These data demonstrate that an IL-6a that is based on an antibody can becreated that has the features of an anti-IL-6 targeting site II of IL-6that can be used to inhibit both cis and trans activity of IL-6, andhave increased PK compared to a parent antibody or other antibody havinga wild type Fc domain effected at least in part by altered binding at pH5.5.

Example 11 Efficacy of Local IL-6 Blockade in Mouse Laser ChoroidalNeovascularization (CNV) Model

To determine whether local IL-6 blockade could be effective for treatingeye disease, e.g., diabetic macular edema (DME) or wet AMD, a monoclonalanti-IL-6 antibody was locally administered in a model system forchoroidal neovascularization. The laser-induced CNV model as describedin Saishin et al. Journal of Cellular Physiology, 195:241-248 (2003) wasemployed in this Example. A laser-induced CNV model reproduces many ofthe pathologic processes underlying diabetic macular edema (DME),including inflammation and angiogenesis.

A monoclonal anti-mouse IL-6 antibody (MP5-20F3, which is a rat IgG1isotype antibody purchased from Bio X Cell, catalog number BE0046) wasadministered to the test group by intravitreal (IVT) injection. Controlsreceived intravitreal injection of VEGF trap or intravitreal injectionof an anti-HRP isotype control antibody (a rat IgG1 against horseradishperoxidase, clone HRPN, purchased from BioXCell; catalog number BE0088).For all antibody groups, 20 μg of protein in a 1 μL volume was injectedinto the test eye, while the contralateral eye was left untreated as afurther control.

Mice were euthalized on day 7 after laser and choroidal flat mounts werestained with Griffonia Simplicifolia (GSA) lectin to measure the lesionarea. FIG. 4 shows the results. The anti-IL-6 antibody treated groupshowed a statistically significant reduction in neovascularizationcompared to the control antibody treated group (p<0.05). On average theanti-IL-6 antibody treated group also showed reduced neovascularizationcompared with the anti-VEGF positive control.

These data demonstrate that an IL-6a, e.g., a monoclonal anti-IL-6antibody, administered IVT can significantly reduce neovascularizationin a mouse CNV model. The results further suggest that an anti-IL-6antibody can produce a reduction in neovascularization at least asgreat, and possibly greater, than an anti-VEGF antibody. These dataindicate that local inhibition of IL-6 is useful for treating eyediseases such as diseases involving vascular leakage, e.g., wet AMD ormacular edema, e.g., diabetic macular edema.

Other embodiments are within the scope of the following claims.

1-36. (canceled)
 37. A isolated antibody or antigen binding fragmentthereof that binds to IL-6 and comprising: a) a human IgG2 isotypeframework comprising one or more amino acid mutations that reduce oreliminate FcRn binding, b) a heavy chain variable (VH) domain comprisingan amino sequence that is at least 95% identical to a VH domain as setforth in amino acids 1-121 of SEQ ID NO: 23, and c) a light chainvariable (VL) domain comprising an amino sequence that is at least 95%identical to a VL domain as set forth in amino acids 1-111 of SEQ ID NO:22.
 38. The antibody or antigen binding fragment of claim 37, wherein aheavy chain comprises the one or more mutations at one or more of aminoacid positions I254, H311, D313, and H346.
 39. The antibody or antigenbinding fragment of claim 38, wherein the one or more mutations compriseone of H311A, H311E, H311N.
 40. The antibody or antigen binding fragmentof claim 38, wherein the one or more mutations comprise D313T.
 41. Theantibody or antigen binding fragment of claim 38, wherein the one ormore mutations comprise one of I254A and I254R.
 42. The antibody orantigen binding fragment of claim 38, wherein the one or more mutationscomprise H436A.
 43. A humanized antibody or antigen binding fragmentthereof, comprising: a) a heavy chain comprising an amino acid sequenceat least 95% identical to SEQ ID NO: 23 comprising zero or moremutations of amino acid positions I254, H311, D313, and H346, whereinCDR sequences in combination differ by no more than 5 amino acids from aVH CDR1 as set forth in SEQ ID NO:4, a VH CDR2 as set forth in SEQ IDNO:5, and VH CDR3 as set forth in SEQ ID NO:6, and b) light chaincomprising CDR sequences that in combination differ by no more than 5amino acids from a VL CDR1 as set forth in SEQ ID NO:7, a VL CDR2 as setforth in SEQ ID NO:8, and a VL CDR3 as set forth in SEQ ID NO:9.
 44. Theantibody or antigen binding fragment of claim 43, wherein the lightchain comprises an amino acid sequence at least 95% identical to SEQ IDNO:
 22. 45. The antibody or antigen binding fragment of claim 44,wherein the one or more mutations comprise one of H311A, H311E, H311N.46. The antibody or antigen binding fragment of claim 44, wherein theone or more mutations comprise D313T.
 47. The antibody or antigenbinding fragment of claim 44, wherein the one or more mutations compriseone of I254A and I254R.
 48. The antibody or antigen binding fragment ofclaim 44, wherein the one or more mutations comprise H436A.
 49. Theisolated antibody or antigen binding fragment of claim 37, comprising a)a VH CDR1 as set forth in SEQ ID NO:4, a VH CDR2 as set forth in SEQ IDNO:5, and VH CDR3 as set forth in SEQ ID NO:6; and b) a VL CDR1 as setforth in SEQ ID NO:7, a VL CDR2 as set forth in SEQ ID NO:8, and a VLCDR3 as set forth in SEQ ID NO:9.
 50. The isolated antibody or antigenbinding fragment of claim 37, wherein the antibody or fragment thereofcomprises: a) CDR sequences that in combination differ by no more than 4amino acids from a VH CDR1 as set forth in SEQ ID NO:4, a VH CDR2 as setforth in SEQ ID NO:5, and VH CDR3 as set forth in SEQ ID NO:6; and b)CDR sequences that in combination differ by no more than 4 amino acidsfrom a VL CDR1 as set forth in SEQ ID NO:7, a VL CDR2 as set forth inSEQ ID NO:8, and a VL CDR3 as set forth in SEQ ID NO:9.
 51. The isolatedantibody or antigen binding fragment of claim 37, wherein the antibodyor fragment thereof comprises: a) CDR sequences that in combinationdiffer by no more than 3 amino acids from a VH CDR1 as set forth in SEQID NO:4, a VH CDR2 as set forth in SEQ ID NO:5, and VH CDR3 as set forthin SEQ ID NO:6; and b) CDR sequences that in combination differ by nomore than 3 amino acids from a VL CDR1 as set forth in SEQ ID NO:7, a VLCDR2 as set forth in SEQ ID NO:8, and a VL CDR3 as set forth in SEQ IDNO:9.
 52. The isolated antibody or antigen binding fragment of claim 37,wherein the antibody or fragment thereof comprises: a) CDR sequencesthat in combination differ by no more than 2 amino acids from a VH CDR1as set forth in SEQ ID NO:4, a VH CDR2 as set forth in SEQ ID NO:5, andVH CDR3 as set forth in SEQ ID NO:6; and b) CDR sequences that incombination differ by no more than 2 amino acids from a VL CDR1 as setforth in SEQ ID NO:7, a VL CDR2 as set forth in SEQ ID NO:8, and a VLCDR3 as set forth in SEQ ID NO:9.
 53. The isolated antibody or antigenbinding fragment of claim 37, wherein the antibody or fragment thereofcomprises: a) CDR sequences that in combination differ by no more than 1amino acid from a VH CDR1 as set forth in SEQ ID NO:4, a VH CDR2 as setforth in SEQ ID NO:5, and VH CDR3 as set forth in SEQ ID NO:6; and b)CDR sequences that in combination differ by no more than 1 amino acidfrom a VL CDR1 as set forth in SEQ ID NO:7, a VL CDR2 as set forth inSEQ ID NO:8, and a VL CDR3 as set forth in SEQ ID NO:9.
 54. A method oftreating a subject having an ocular disease characterized by an elevatedlevel of IL-6 in the vitreous, the method comprising administering tothe subject the antibody or antigen binding fragment of claim
 49. 55.The method of claim 54, wherein the ocular disease is selected from thegroup consisting of diabetic macular edema (DME), diabetic retinopathy,uveitis, dry eye syndrome, uveitis, age-related macular degeneration(AMD), proliferative diabetic retinopathy (PDR), retinal vein occlusion(RVO), neuromyelitis optica (NMO), corneal transplant, corneal abrasion,and physical injury to the eye.
 56. The method of claim 55, wherein theocular disease is DME.
 57. The method of claim 56, wherein the antibodyor antigen binding fragment is delivered to the vitreous of thesubject's eye.
 58. The antibody or antigen binding fragment of claim 37,wherein the antibody exhibits reduced FcRn binding compared to acorresponding antibody having a human wild type Fc domain.
 59. Theantibody or antigen binding fragment of claim 43, wherein the antibodyexhibits reduced FcRn binding compared to a corresponding antibodyhaving a human wild type Fc domain.